Methods and compositions for treating genetically linked diseases of the eye

ABSTRACT

Expression vectors and therapeutic methods of using such vectors in the treatment of diseases of the eye resulting from failure to produce a specific protein in the eye, or the production of a non-functional protein in the eye.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority under 35 U.S.C. §119(e)to U.S. Provisional Patent Application Ser. No. 61/765,654 filed Feb.15, 2013 and U.S. Provisional Patent Application Ser. No. 61/815,636filed Apr. 24, 2013, both of which are incorporated herein by reference.

TECHNICAL FIELD

The invention relates to gene therapy and specifically, expressionvectors and therapeutic methods of using such vectors in the treatmentof diseases of the eye resulting from failure to produce a specificprotein in the eye, or the production of a non-functional protein in theeye.

BACKGROUND OF INVENTION

Several diseases of the eye result from an underlying genetic cause. Forexample, in some diseases, a mutation in a protein expressed in cells ofthe eye alters, or abolishes, the proteins activity resulting in adisease state. In other diseases, the cause may be due to failure of eyecells to produce a particular protein. Because these diseases are due toinactivation, or alteration, of a single protein they are particularlyamenable to gene transfer-based therapies. Gene therapy for oculardisease has a set of attractive attributes, including a small tissuetarget and a closed compartment, which thereby requires a low dose.Additionally the eye is a relatively immune-privileged environment.

One example of an eye disease having a genetic cause is X-linkedjuvenile retinoschisis (XLRS). XLRS is a neurodevelopmental retinalabnormality that manifests early in life and causes impaired acuity anda propensity to retinal detachment. XLRS is characterized by structuralabnormalities in normal lamination of the retinal neuronal and plexiformlayers. Clinical examination shows microcysts within the macula, andschisis or internal dissection of the layers of the peripheral retina,(Eksandh L C, Ponjavic V, Ayyagari R, Bingham E L, Hiriyanna K T,Andreasson S, Ehinger B, Sieving P A. 2000. Phenotypic expression ofjuvenile X-linked retinoschisis in Swedish families with differentmutations in the XLRS1 gene. Arch Ophthalmol 118: 1098-1104; Prenner JL, Capone A, Jr., Ciaccia S, Takada Y, Sieving P A, Trese M T. 2006.Congenital X-linked retinoschisis classification system. Retina 26:S61-64) and this is evident by using ocular coherence tomography (GerthC, Zawadzki R J, Werner J S, Heon E. Retinal morphological changes ofpatients with X-linked retinoschisis evaluated by Fourier-domain opticalcoherence tomography. Arch Ophthalmol. 2008; 126:807-11). Impairedretinal synaptic transmission of neural signals causes loss ofdark-adapted absolute visual perception. This is evident on clinicalelectroretinogram (ERG) testing as a characteristic reduction of theb-wave response (from second-order retinal bipolar cells) relative tothe photoreceptor a-wave, which frequently gives rise to an‘electronegative ERG waveform.’ The fragile XLRS retina is more prone todisease related complications, such as vitreous hemorrhage and retinaldetachment, and the condition worsens with age. The rate of retinaldetachment in the XLRS population is considerably higher than in thegeneral population (10 vs 0.01%, respectively), and the postoperativeoutcome is much worse.

X-linked juvenile retinoschisis is caused by mutations in thegene-encoding retinoschisin, a 224-amino acid secreted protein that isexpressed only by the retina and pineal. Human retinoschisin is composedof a 23-amino acid signal sequence, a 39-amino acid Rs1 domain, a157-amino acid discoidin domain and a 5-amino acid C-terminal segment.Discoidin domain containing proteins are widely distributed ineukaryotes and mediate a variety of functions, including cell adhesion,cell-extracellular matrix interactions, signal transduction,phagocytosis of apoptotic cells, axon guidance, angiogenesis and bloodclotting. Many of these proteins are involved in extracellular matrix orcell binding, although some bind ligands such as vascular endothelialgrowth factor and semaphorin. Retinoschisin is secreted from retinalneurons as a disulfide-linked homo-octameric complex, which adheres tothe cell surface, but its function is not well understood. Biochemicalactivities attributed to retinoschisin are the binding of b-2-laminin,ab-crystallin, phospholipid, galactose and Na/K ATPase-SARM1 complex.Retinoschisin is first observed in the mouse retina on postnatal day 1.During development, all retinal neurons express retinoschisin afterdifferentiation, beginning with the ganglion cells, which are the firstto mature, followed by neurons of each of the more distal layers. FromP14 onward, it is strongly expressed in the outer half of the innernuclear layer and by photoreceptor inner segment. All classes of retinalneurons, except horizontal cell, are shown to be labeled withretinoschisin antibody in adults.

Multiple groups have attempted to use gene-therapy approaches for thetreatment of diseases of the eye. For example, several groups have usedadeno-associated virus (AAV) vectors expressing retinoschisin tocomplement the mutations of mice harboring retinoschisin gene deletions.Retinal transduction with these vectors resulted in significant levelsof retinoschisin protein in all layers of the retina, and improvement ofthe disease phenotype, including restoration of the normal positive ERGb-wave and a reduction of the cyst-like structures that arecharacteristic of the disease. The therapeutic effect was durable andpersisted throughout the life of the animal.

In addition to the treatment of X-linked retinoschinosis, other groupshave evaluated the clinical use of AAV vectors for the treatment ofanother X-linked retinopathy, Leber congenital amaurosis (LCA), becauseof congenital retinal pigment epithelium (RPE) 65 deficiency. AAVvectors expressing RPE65 were administered by subretinal injection to atotal of nine subjects with LCA. The nine subjects comprised thecollective low-dose cohorts of the three studies, each of which have adose-escalation design. The majority of the treated subjects showedevidence of improvement in retinal function, visual acuity or reductionin nystagmus despite their relatively advanced state of retinaldegeneration.

While the LCA trials used subretinal injection to deliver the vector,this delivery strategy may be problematic for an XLRS trial, assubretinal injection gives geographically localized delivery.Retinoschisin is expressed throughout the retina and optimal treatmentof the disease will require transduction of the entire retina. Vectordelivery by subretinal injection is limited maximally to about 25% ofthe retinal area. Although this amount of transduction is sufficient tocover the vicinity of the macula, much of the retina would probably notbe transduced, and the untreated area would remain susceptible toretinal detachment and vitreous hemorrhage, which are the major causesof vision loss with this disease. Some additional spread ofretinoschisin has been reported in retinas of mice transduced bysubretinal injection, but it is not clear how this might scale to humansubjects. Subretinal injection of retinas with schisis pathology may bechallenging and pose a significant risk to the visual function of thesubject. Vitrectomy is usually carried out before subretinal injection.Adhesion of the vitreous to the retina may cause further laminarsplitting of the fragile XLRS retina when the surgeon attempts toseparate the vitreous from the retina. In addition, the injection itselfmay also be difficult. If the tip of the injection needle is notpositioned deep enough, vector solution may be inadvertently routed intothe schisis cavities and exacerbate the intraretinal splitting. Analternative vector administration method would be attractive for XLRSsubjects.

In previous work, the inventors described a method for obtainingefficient AAV vector-mediated gene transfer to XLRS retinas without theuse of subretinal injection. In that study, all layers of theretinoschisin knockout (Rs1-KO) mouse retina were efficiently transducedwith AAV type 2 (AAV2) vectors when administered by simple vitreousinjection (Zeng Y, Takada Y, Kjellstrom S, Hiriyanna K, Tanikawa A,Wawrousek E, et al. RS-1 Gene Delivery to an Adult Rs1h Knockout MouseModel Restores ERG b-Wave with Reversal of the Electronegative Waveformof X-Linked Retinoschisis. Invest Ophthalmol Vis Sci. 2004; 45:3279-85;Kjellstrom S, Bush R A, Zeng Y, Takada Y, Sieving P A. Retinoschisingene therapy and natural history in the Rs1h-KO mouse: long-term rescuefrom retinal degeneration. Invest Ophthalmol Vis Sci. 2007; 48:3837-45).However, administration of AAV2 vector leads to a therapy-limitingimmune response in the eye, since humans have a high preexistingimmunity to AAV2. The inventors developed an AAV vector to complementvitreal administration in humans. The vector was composed of a 3.5-kbhuman retinoschisin promoter, a human retinoschisin cDNA containing atruncated retinoschisin first intron, the human b-globin polyadenylationsite and AAV type 2 (AAV2) inverted terminal repeats, packaged in an AAVtype 8 capsid. Intravitreal administration of this vector to Rs1-KO miceresulted in robust retinoschisin expression with a retinal distributionthat was similar to that observed in wild-type retina. Immunolabelingwas specific to the retinoschisin-expressing cells of the retina withlittle or no off-target expression in other eye structures, such as theoptic nerve, uveal tissue and cornea.

Thus, the present invention addresses the need for an improved method ofdelivering therapeutic molecules, such as genes encoding therapeuticproteins, to the eye of an individual in need of such treatment, withouteliciting a significant immune response, and provides other benefits aswell.

SUMMARY OF THE DISCLOSURE

We have surprisingly found that the inventive compositions and methodsof administration are capable of inducing the production of proteins intissues of the eye while minimizing or avoiding unwanted inflammatoryresponses or other unwanted side effects. Thus, the invention providesexpression vectors and therapeutic methods of using such vectors in thetreatment of diseases of the eye, particularly disorders of the eyeresulting from failure to produce a specific protein in the eye, or theproduction of a non-functional protein in the eye.

One embodiment of the present invention is a method of treating anindividual having a disease of the eye, the method comprisingadministering to the individuals eye a vector comprising a nucleic acidsequence encoding a therapeutic protein, wherein the expression vectorexpresses a high level of the therapeutic protein, and whereinadministration of the viral vector elicits a minimal immune response. Inone embodiment, administration of the vector does not elicit atherapy-limiting immune response within the individual. The nucleic acidencoding the therapeutic protein may be linked to an eye-specificpromoter. Further, the promoter may be specific for certain portions ofthe eye, such as the retina. In such embodiments, a retina-specificpromoter may comprise a portion of a retinoschisin gene promoter. In oneembodiment, the retina-specific promoter comprises at least a portion ofSEQ ID NO:9.

In certain embodiments, genetic elements, such as enhancer elements, maybe included to enhance expression of the therapeutic protein. In oneembodiment, the therapeutic protein is linked to a promoter comprisingan interphotoreceptor retinoid-binding protein (IRBP) enhancer sequence.In one embodiment, the IRBP enhancer sequence comprises SEQ ID NO:12.

Methods of the present invention are useful for treating diseases of theeye. In one embodiment, the disease of the eye is selected from thegroup consisting of retinoschisis, age-related macular degeneration(AMD), diabetic retinopathy, Leber congenital amaurosis (LCA), retinaldetachment (due to disease, injury or spontaneous detachment), cysts,cystoid macular edema, retinitis pigmentosa, and senile schisis. In oneembodiment, the disease of the eye is linked with the x-chromosome.

In one embodiment, the therapeutic protein is a retinoschisin protein.The retinoschisin protein may have at least 90% sequence identity to thesequence of a known retinoschisin protein or any portion thereof. Forexample, the retinoschisin protein may have at least 90% sequenceidentity to SEQ ID NO:2, or any portion thereof, so long as the proteinencoded by a vector of the present invention has at least one activityof a wild-type retinoschisin protein. In one embodiment, a nucleic acidsequence encoding a retinoschisin protein of the present inventioncomprises at least one splice donor and one lariat/splice acceptor site.The splice donor and the lariat/splice acceptor site may be from intron1 of a retinoschisin gene. The nucleic acid sequence encoding thetherapeutic protein may also be linked to a polyadenylation signal, suchas the human beta-globin 3′ polyadenylation signal.

In one embodiment of the present invention, the vector comprisesadeno-associated virus inverted terminal repeats (ITRs). The ITRs may ormay not be identical in sequence. One of the ITRs may lack the REPprotein nicking recognition sequence or the D region. At least one UTRmay be derived from, or may consist of, the psub201 vector. In oneembodiment, the vector comprises SEQ ID NO:16. In one embodiment, thevector comprises capsid proteins from one or more adeno-associatedviruses. In one embodiment, the capsid protein is from AAV8. In oneembodiment, the vector is administered by intravitreal injection.

One embodiment of the present invention is an expression vectorcomprising a nucleic acid sequence encoding a therapeutic protein,wherein the expression vector expresses a high level of the therapeuticprotein when administered to the eye of an individual. Vectors of thepresent invention elicit a minimal immune response in the individualwhen administered to the eye. Further, vectors of the present inventionalleviate at least one symptom of retinoschisis when administered to theeye at a dose that elicits an insignificant immune response. Viralvectors useful for treating diseases of the eye may be prepared byincubating expression vectors of the present invention with cellsexpressing AAV capsid proteins and AAV REP proteins. The AAV capsid andREP proteins may be provided by a plasmid, by a helper virus or by genesintroduced into the genome of the cells.

One embodiment of the present invention is an expression vector for usein ocular gene therapy applications comprising: a capsid protein thathas low preexisting immunity in humans; an expression cassette thatproduces a therapeutic level of protein in the individual whenadministered to the individual at a dose that does not elicit atherapy-limiting immune response within the individual followingadministration by intravitreal injection; and, a tissue-specificpromoter that inhibits or prevents expression of the expression vectorin antigen presenting cells and/or tissues that do not normally expressthe therapeutic protein. In one embodiment, the immune response producedwithin the individual following administration by intravitreal injectionis less than or equal to +2 cells transiently and +1 cells chronically.Further, expression in antigen presenting cells and tissues outside ofthe eye is less than 1% of expression in a tissue of the eye. In oneembodiment, the tissue-specific promoter is a retina-specific promoter.The tissue-specific promoter may comprise at least a portion of SEQ IDNO:9. The expression vector may also comprise adeno-associated virus(AAV) inverted terminal repeat (ITR) sequences. In one embodiment, theexpression vector comprises SEQ ID NO:16. The expression vector may alsocomprise capsid proteins from one or more adeno-associated viruses. Inone embodiment, the expression vector comprises capsid proteins fromAAV8.

One embodiment of the present invention is a method of treating anindividual having a disease of the eye, comprising administering to thepatient's eye an expression vector comprising a capsid protein that haslow preexisting immunity in humans; an expression cassette that producesa therapeutic level of protein in the individual when administered tothe individual at a dose that does not elicit a therapy-limiting immuneresponse within the individual following administration by intravitrealinjection; and, a tissue-specific promoter that inhibits or eliminatesexpression of the expression vector in antigen presenting cells andtissues that do not normally express the therapeutic protein. In oneembodiment, the immune response produced within the individual followingadministration by intravitreal injection is less than or equal to +2cells transiently and +1 cells chronically. Further, expression inantigen presenting cells and tissues outside of the eye is less than 1%of expression in a tissue of the eye. In one embodiment, thetissue-specific promoter is a retina-specific promoter. Thetissue-specific promoter may comprise at least a portion of SEQ ID NO:9.The expression vector may also comprise adeno-associated virus (AAV)inverted terminal repeat (ITR) sequences. In one embodiment, theexpression vector comprises SEQ ID NO:16. The expression vector may alsocomprise capsid proteins from one or more adeno-associated viruses. Inone embodiment, the cassette comprises capsid proteins from AAV8.

One embodiment of the present invention is a method of treating X-linkedretinoschisis in a human comprising: administering to a human subjectdiagnosed with, or suspected of having, X-linked retinoschisis atherapeutically effective amount of an expression vector comprising: acapsid protein from AAV8; an expression cassette comprising aretinoschisin gene promoter operably linked to an interphotoreceptorretinoid-binding protein (IRBP) enhancer sequence, adeno-associatedvirus (AAV) inverted terminal repeat (ITR) sequences, and a humanretinoschisin protein, wherein administration of the expression vectorcauses expression of the human retinoschisin protein in a retinal cellof the subject, and reduces at least one symptom of retinoschisis. Theexpression vector may be administered using intravitreal, subretinal orsubtenon injection techniques. The expression vector may also beadministered topically.

The expression vector of the invention is administered in an amount thatis therapeutically effective. A therapeutically effective amountincludes, for example, a dose between about 1e¹⁰ vg/eye to about 2.5e¹¹vg/eye, a dose between about 1e⁸ vg/eye to about 1e¹³ vg/eye, a dosebetween about 1e⁹ vg/eye to about 1e¹³ vg/eye, a dose between about 3e⁹vg/eye to about 1e¹³ vg/eye, a dose between about 1e¹⁰ vg/eye to about1e¹³ vg/eye, a dose between about 3e¹⁰ vg/eye to about 1e¹³ vg/eye, adose between about 1e¹¹ vg/eye to about 1e¹³ vg/eye, a dose betweenabout 3e¹¹ vg/eye to about 1e¹³ vg/eye.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the structure of vector pAAV scRS/IRBP hRS.

FIG. 2 shows the AAV8 hRS/IRBP and AAV8 hRSp4 vectors evaluated onCoomassie 8250 (panel A) and silver stained (panel B) 7.5% SDS gels.Sample order:

left panel (Coomassie): Standard, AAV8 hRS/IRBP, AAV8 hRSp4, Standard;

right panel (silver): Standard, AAV8 hRS/IRBP, AAV8 hRSp4.

All vector loaded at 2e10 vg/lane. Standards are (from the top): 250kd,150kd, 100kd, 75kd, 50kd, 37kd, and 25kd.

FIG. 3 shows OCT scans from a wild type and an Rs1-KO mouse showing aB-scan (left-hand images) taken through the central retina at the opticnerve as indicated by the central green line on the volume intensityprojection (right-hand images) for each eye.

FIGS. 4A and 4B show the scoring of retinoschisin immunostaining in AAV8scRS/IRBP hRS treated retinas of Rs1-KO mice.

FIG. 5 shows the ERG a- and b-wave amplitudes of untreated eyes and eyestreated with intravitreal injections of vehicle or AAV8 scRS/IRBP hRSvector at various doses. The eyes were evaluated between 11 and 15 weekspost injection.

FIG. 6 shows the ERG a- and b-wave amplitudes in animals receiving 1e8,5e8 and 2.5e9 vg/eye vector doses at 6-9 months post injection.

FIG. 7 presents a comparison of the Short Term and Long Term ERG resultsfor treated and untreated eyes derived from the data sets shown in FIGS.5 and 6.

FIG. 8 shows the schisis cavity scoring averages in treated anduntreated eyes from OCT images for various vector doses.

FIG. 9 depicts retinoschisin protein expression in response to vectordoses between 1e7, and 2.5e9 vg/eye.

FIG. 10 depicts ophthalmological findings in New Zealand White rabbitsinjected intravitreally with 2 doses of either vehicle (A) or AAV8scRS/IRBP hRS 2e¹⁰ vg/eye (B) or 2e¹¹ vg/eye (C).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention generally relates to improved methods for treatingdisorders of the eye, including, for example, x-linked retinoschisis(XLRS), retinal detachment (disease-related, injury-induced, andspontaneous), age-related macular degeneration, cysts, cystoid macularedema, retinitis pigmentosa, and senile schisis, as well as vectorsuseful in such treatment methods. More specifically, the presentinvention relates to an improved expression vector that is able toeffect high level expression of an encoded protein in an eye, withminimal elicitation of an immune response. Because of thesecharacteristics, such vectors are particularly useful for treatingdiseases of the eye that result from either failure to produce aspecific protein in the eye, or the production of a non-functionalprotein in the eye.

A method of the present invention can generally be accomplished byadministering to the eye of a patient in need of such treatment, anexpression vector that expresses high levels of a therapeutic moleculein the eye, wherein the administration of the expression vector eitherfails to elicits an immune response, or elicits a minimal immuneresponse in the eye of the treated patient.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. For example, a nucleic acid moleculerefers to one or more nucleic acid molecules. As such, the terms “a”,“an”, “one or more” and “at least one” can be used interchangeably.Similarly the terms “comprising”, “including” and “having” can be usedinterchangeably. It is further noted that the claims may be drafted toexclude any optional element. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology as“solely,” “only” and the like in connection with the recitation of claimelements, or use of a “negative” limitation

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates, which may need to be independently confirmed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodiments arespecifically embraced by the present invention and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed. In addition, all sub-combinations are also specificallyembraced by the present invention and are disclosed herein just as ifeach and every such sub-combination was individually and explicitlydisclosed herein.

As used herein, the terms individual, subject, and patient arewell-recognized in the art, and are herein used interchangeably to referto any human or other animal in need of treatment of a disease of theeye. Examples include, but are not limited to, humans and otherprimates, non-human primates such as chimpanzees and other apes andmonkey species; farm animals such as cattle, sheep, pigs, seals, goatsand horses; domestic mammals such as dogs and cats; laboratory animalsincluding rodents such as mice, rats and guinea pigs; birds, includingdomestic, wild and game birds such as chickens, turkeys and othergallinaceous birds, ducks, geese, and the like. A preferred patient totreat is a human patient. The terms individual, subject, and patient bythemselves, do not denote a particular age, sex, race, and the like.Thus, individuals of any age, whether male or female, are intended to becovered by the present disclosure and include, but are not limited tothe elderly, adults, children, babies, infants, and toddlers. Likewise,the methods of the present invention can be applied to any race,including, for example, Caucasian (white), African-American (black),Native American, Native Hawaiian, Hispanic, Latino, Asian, and European.

The present invention can be used to treat any disease of the eye inwhich the disease results from either inappropriate expression of aprotein, or expression of a malfunctioning or dysfunctional form of aprotein expressed in the eye. Inappropriate expression of a protein mayrefer to lack of expression, under-expression or over-expression of aprotein. Expression of a malfunctioning form of a protein refers toexpression of a protein having one or more mutation(s) that alters theactivity of the protein. Alteration of activity may refer to completeinactivation of protein activity, reduction of protein activity or anincrease in protein activity. Altered activity may result from, forexample, direct inactivation of an active site or misfolding of theprotein. Examples of eye diseases that may be treated using the presentinvention include, but are not limited to X-linked retinoschisis,age-related macular degeneration (AMD), diabetic retinopathy, Lebercongenital amaurosis (LCA), retinal detachment (due to disease, injury,or spontaneous), cysts, cystoid macular edema, retinitis pigmentosa, andsenile schisis. Thus, the expression cassette of the invention mayinclude, for example, polynucleotide sequences encoding proteins such asciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor(BDNF), pigment epithelium-derived factor (PEDF), or pigmentepithelium-derived factor (PEDF). For example, the inventors have testeda vector expressing lens epithelial derived growth factor (LEDGF) anddemonstrated a protective effect in the RCS rat model of Retinitispigmentosa (RP).

In a preferred embodiment, the eye disease treated is X-linkedretinoschisis. X-linked retinoschisis is a neurodevelopmental retinalabnormally that causes impaired acuity and a propensity to retinaldetachment. XLRS is characterized by structural abnormalities in normallamination of the retinal neuronal and plexiform layers. Clinicalexamination shows microcysts within the macula, and schisis or internaldissection of the layers of the peripheral retina. X-linked juvenileretinoschisis is caused by mutations in the gene encoding retinoschisin,a 224-amino acid, secreted protein that is expressed only by the retinaand pineal.

As used herein, an expression vector is a recombinant nucleic acidmolecule comprising a nucleic acid sequence (e.g., open-reading frame(ORF)) that encodes a therapeutic molecule of the present invention,wherein the nucleic acid sequence is linked to a promoter that driveshigh level expression of the therapeutic molecule when the expressionvector is administered to, for example, a subject or an organ, tissue orcell. An expression vector of the present disclosure is produced byhuman intervention and can be DNA, RNA or variants thereof. Theexpression vector may be a linear molecule (e.g., a linear nucleic acidmolecule, a linear viral genome, etc.) or it may be a circular moleculesuch as, for example, a plasmid. In one embodiment, an expression vectormay comprise one or more nucleic acid sequences from an adeno-associatedvirus (an AAV vector), a cytomegalovirus (CMV) (a CMV vector), aretrovirus, an adenovirus, a herpes virus, a vaccinia virus (a vacciniavector), a poliovirus, a Sindbis virus, or any other DNA or RNA virus.In one embodiment, an expression vector may be a DNA plasmid. In oneembodiment, an expression vector may be a viral genome. In oneembodiment, an expression vector may be a DNA molecule, either linear orcircular, comprising nucleic acid sequences from a plasmid and nucleicacid sequences from a viral genome to enable nucleic acid moleculedelivery and high-level expression of the encoded therapeutic molecule.In one embodiment, the expression vector is an AAV expression vector. Asused herein, an AAV expression vector is a nucleic acid moleculecomprising AAV sequences that allow for the replication, packagingand/or expression of the nucleic acid molecule. General methods for theconstruction of expression vectors are known in the art, and are alsodisclosed in, for example, Molecular Cloning: a Laboratory Manual,3^(rd) edition, Sambrook et al. 2001 Cold Spring Harbor LaboratoryPress, and Current Protocols in Molecular Biology, Ausubel et al. eds.,John Wiley & Sons, 1994, both of which are incorporated herein byreference in their entirety.

As noted above, expression vectors of the present invention comprisepromoters that drive high-level expression of nucleic acid sequencesencoding therapeutic molecules. As used herein, the phrase “driveexpression” refers to the ability of a promoter to promote transcriptionfrom an open reading frame (ORF). According to the present disclosure,promoters used in expression vectors of the present invention arespecific for cells of the eye (i.e., eye-specific promoters). That is,the promoter only drives expression from the ORF when the expressionvector is introduced into a cell of the eye. Such promoters are specificfor cells such as, photoreceptor cells, bipolar cells, horizontal cells,amacrine cells, ganglion cells, rods and cones. Examples of suchpromoters include, but are not limited to, a retinoschisin promoter, arhodopsin promoter, a rhodopsin kinase promoter, a CRX promoter, and aninterphotoreceptor retinoid binding protein (IRBP) promoter. Anypromoter that allows eye-specific expression of an encoded protein canbe used, so long as the promoter drives high-level expression of theORF. Thus, in one embodiment, the expression vector comprises aneye-specific promoter.

As used herein, the phrase high-level expression refers to the abilityof vectors (i.e., expression vectors and viral vectors comprisingexpression vectors) of the present invention to express the therapeuticmolecules at levels high enough such that the amount of vector requiredto alleviate symptoms of the eye disease elicits a minimal, or no,immune response. According to the present invention, alleviation ofsymptoms of eye disease refers to the ability of a therapeutic moleculeto reduce, or eliminate, the pathology, and the related symptoms, froman eye disease. Such alleviation may completely eliminate symptoms ofeye disease and restore the patients' eye to a normal level offunctioning, or it may reduce some of the pathology and restore partialfunction to the patient's eye. It is understood by those skilled in theart that normal and partial levels of function are relative terms, andare determined by comparing the level of function in the treated eyewith the level of function observed in the eyes of a comparable cohortof individuals (e.g., individuals of the same age, race, sex, etc.).Methods of determining the level of eye function, in an individual areknown to those skilled in the art. It is also understood by thoseskilled in the art that determining the levels of therapeutic moleculeneeded may be an empirical process. However, once such levels are known,they can be quantified by comparing the levels to the levels ofexpression observed using a reference promoter. Once such a referencehas been established, high-level expression may refer to the ability ofa promoter to cause expression of an ORF at levels that aresignificantly higher than the level of expression observed using thereference promoter. An example of a reference promoter is described byColosi et al. (Gene Therapy, 16, 2000, 916-926). In one embodiment,promoters of the present invention may cause transcription of ORFs at alevel that is at least 5×, 10×, 20×, 50×, 100×, 500× or at least 1000×higher than a reference promoter. Levels of expression can be comparedby, for example, comparing the level of ORF-specific mRNA produced eachexpression vector. Methods of performing such comparisons are known tothose skilled in the art.

As used herein, a minimal immune response refers to an immune responsegenerated against a construct (e.g., a vector) of the present inventionthat is not therapeutically limiting. Thus, for example, whileconstructs of the present invention may elicit an immune response, theimmune response is manageable using standard medical practices, such asthe administration of steroidal or non-steroidal anti-inflammatorycompounds/compositions. Such an immune response may also be referred toas resolvable. In one embodiment, administration of the vector fails toelicit any immune response against the vector. In another embodiment,administration of the vector fails to elicit a therapy-limiting immuneresponse against the vector. In another embodiment, administration ofthe vector fails to elicit a dosage-limiting immune response against thevector. In another embodiment, administration of the vector fails toelicit a detectable immune response against the vector. In anotherembodiment, administration of the vector elicits only atherapeutically-manageable immune response against the vector. Inanother embodiment, less than 50% of the vector is neutralized byintravenous immune globulin (IVIG) at 20 mg/ml in a vectorneutralization assay (see, Arbetman, A. E., et al., Novel CaprineAdeno-Associated Virus (AAV) Capsid (AAV-Go.1) Is Closely Related to thePrimate AAV-5 and Has Unique Tropism and Neutralization Properties, JVirol. 2005 December; 79(24):15238-15245). In another embodiment, theimmune response produced within the individual following administrationof the vector is less than or equal to +2 cells transiently and +1 cellschronically.

One type of tissue-specific promoter is an eye-specific promoter. Forexample, a promoter that drives expression of a ORF only when it is in acell of the retina (including, for example, bipolar cells, horizontalcells, amacrine cells, ganglion cells, rods and cones) is referred to asa retina-specific promoter. Thus, in one embodiment, the promoter is aretina-specific promoter. In one embodiment, the expression of the viralvector containing an eye-specific promoter in antigen presenting cellsand tissues outside of the eye is less than 1% of expression in a tissueof the eye.

One example of a retina-specific promoter is the retinoschisin genepromoter, the sequence of which is represented by SEQ ID NO:9. WithinSEQ ID NO:9, bases 1-8 are the engineered NotI site for cloning; bases9-248 human RS promoter sequence; bases 249-254 are the engineered SalIsite for addition of IRBP enhancer; bases 255-515 are the human IRBPenhancer sequence; bases 516-521 are the engineered SalI site foraddition of IRBP enhancer; bases 522-750 are the proximal retinoschisinpromoter; bases 551-802 are the retinoschisin 1st exon; bases 803-1063are the splice donor and proximal retinoschisin 1st intron; bases1064-1071 are the engineered AscI site for ligation to splice acceptorof intron.

Intron sequences are included in the promoter because they increase mRNAexport from the nucleus to the cytoplasm compared to an intron-lessconstruct for most cDNAs, resulting in an approximately 10-fold increasein transgene expression. Viruses have evolved other mechanisms tofacilitate the export of viral mRNAs that don't involve splicing. Byinhibiting splicing, these viruses can divert protein production fromhost mRNAs to viral mRNA late in viral replication. Elements thatviruses use to accomplish this mRNA transport include the WPRE(Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element), andRRE (HIV and SIV rev response element).

Thus, in one embodiment, the promoter comprises at least a portion of aretinoschisin promoter. In a specific embodiment, the portion is aportion of SEQ ID NO:9. In one embodiment, the promoter comprises anucleotide sequence that is at least 95% identical to at least onesequence selected from the group consisting of SEQ ID NO:10 and SEQ IDNO:11, wherein the promoter has retinoschisin gene promoter activity. Inone embodiment, the promoter comprises at least one sequence selectedfrom the group consisting of SEQ ID NO:10 and SEQ ID NO:11, wherein thepromoter has retinoschisin gene promoter activity. In one embodiment,the promoter comprises SEQ ID NO:9. In one embodiment, the promotercomprises SEQ ID NO:9. In one embodiment, the promoter consists of SEQID NO:9.

The present inventors have discovered that modifications to promoters,such as the retinoschisin promoter, may result in significantimprovement in the ability of the promoter to drive expression of anORF. Examples of modifications that may be useful for improving theperformance of promoters of the present invention include sequencemutations (e.g., nucleotide substitutions, additions, or deletions), andthe addition, or removal, of regulatory elements, such as transcriptionfactor binding elements, enhancer elements, silencer elements andboundary elements. Examples of such elements include a TATA element, a Brecognition element, and an E-box element. Thus, in one embodiment, theeye-specific promoter has been modified so that it comprisesheterologous nucleic acid sequences. As used herein, heterologousnucleic acid sequences are nucleic acid sequences that, in their naturalsetting (e.g., in a genome) are not linked to the sequences to whichthey are being referenced. For example, with regard to eye-specificpromoters present in expression vectors of the present invention,sequences that are heterologous thereto are any nucleic acid sequencesnot found in association with such eye-specific promoter sequences incells of the eye. Preferably, any elements added to the promoter regionare specific to cells of the eye. In one embodiment, an expressionvector of the present invention comprises a promoter comprising anenhancer element. One example of a useful enhancer element is aninterphotoreceptor retinoid binding protein (IRBP) enhancer element,which is represented by SEQ ID NO:12. In one embodiment, the promotercomprises at least a portion of the IRBP promoter. In one embodiment,the enhancer element comprises a nucleotide sequence at least 95%identical to SEQ ID NO:12, wherein the enhancer retains the ability toenhance transcription from a nearby promoter (i.e., a promoter within500 nucleotides of either end of the enhancer sequence). In oneembodiment, the IBRP enhancer element comprises SEQ ID NO:12. In oneembodiment, the IRBP enhancer element is linked to one end of theeye-specific promoter. In one embodiment, the IRBP enhancer element isinserted within the sequence of the eye-specific promoter. In oneembodiment, the IRBP enhancer element is inserted within the sequence ofthe retinoschisin gene promoter.

As used herein, a therapeutic molecule is a molecule that whenintroduced within the eye, is capable or ameliorating or eliminatingsymptoms of a disease of the eye. Examples of therapeutic moleculesinclude proteins and RNAs, including siRNAs. Such molecules may act byproviding an activity that is missing, or significantly reduced, in adiseased eye. Such molecules may also act by modifying or reducing anactivity that is over-expressed, or significantly elevated above normallevels, in a diseased eye. For example, a therapeutic molecule may be aprotein possessing an activity (e.g., specific binding activity,enzymatic activity, transcriptional regulation activity, etc.) that islacking in cells of the eye. Lack of such activity may result fromfailure of the cells to produce the protein, production of a mutated,inactive form of the protein, or misfolding of a protein resulting in aninactive form. In some cases, introducing a “good” (i.e., functional)copy of the protein may alleviate symptoms of the disease by directlyreplacing the missing activity. Alternatively, therapeutic molecules mayact by increasing or decreasing the activity of other proteins in cellsof the eye. For example, the therapeutic protein may bind to anotherprotein and thereby either decrease, or eliminate the activity of thesecond protein. Alternatively, binding of the therapeutic protein toanother protein in cells of the eye may result in stabilization of suchprotein and/or an increase in the related activity. Finally, thetherapeutic molecule may increase or decrease transcription of genes, orthe translation of transcripts from genes in cells of the eye. Forexample, a therapeutic protein may bind to a transcriptional region of agene and thereby increase or decrease transcription of that gene.

Any protein can be used as a therapeutic protein, provided the proteinpossesses an activity that is of therapeutic benefit in treating adisease of the eye. For example, if the disease to be treated is relatedto abnormal blood vessel growth (e.g., wet, age-related, maculardegeneration (AMD), diabetic retinopathy, etc.) a useful therapeuticprotein could be any protein having anti-angiogenic activity. As afurther example, if the disease to be treated is due to neuropathy inthe eye (e.g., glaucoma, retinitis pigmentosa, etc.) a usefultherapeutic protein, may be any protein, or molecule, that provides aneuroprotective effect in the eye. Examples of such proteins include,but are not limited to, ciliary neurotrophic factor (CNTF),brain-derived neurotrophic factor (BDNF) and pigment epithelium-derivedfactor (PEDF).

One example of a useful therapeutic protein is retinoschisin protein,which is a 224-amino acid, discoidin domain-containing, retina-specific,secretory protein. Loss of retinoschisin protein function has beenimplicated in X-linked retinoschinosis. As used herein, a retinoschisinprotein refers to a full-length retinoschisin protein, or any portionthereof, that has at least one activity of a wild-type retinoschisinprotein. Thus, in one embodiment, the therapeutic protein comprises atleast a portion of a retinoschisin protein. Such a portion may compriseat least 50 amino acids, at least 75 amino acids, at least 125 aminoacids, at least 150 amino acids, at least 175 amino acids or at least200 amino acids, so long as the resulting therapeutic protein possessesat least one function of a full length retinoschisin protein. In arelated embodiment, the therapeutic protein is a retinoschisin proteinhaving at least 90%, at least 95%, at least 97%, at least 98% or atleast 99% sequence identity to a full-length retinoschisin protein, orany portion thereof, that has at least one activity of a wild-typeretinoschisin protein. In a specific embodiment, the therapeutic proteinis a human retinoschisin protein having at least 90%, at least 95%, atleast 97%, at least 98% or at least 99% sequence identity to afull-length human retinoschisin protein (SEQ ID NO:2 or SEQ ID NO:5), orany portion thereof, that has at least one activity of a wild-typeretinoschisin protein. Known functions of the retinoschisin proteininclude binding to anionic phospholipids, binding to the sterile alphaand TIR motif-containing protein (SARM1), binding to alpha-B crystallineprotein and binding to beta2 laminin.

As noted above, the retinoschisin protein comprises a discoidin domain,a structure that has been found in other secreted and transmembraneproteins. While the function of the discoidin domain in theretinoschisin protein is not well understood, in other proteins it hasbeen implicated in cell-cell adhesion and cell-cell signaling. Withregard to retinoschisin, it has been demonstrated that introduction ofmutations that alter the discoidin domain structure result in loss ofretinoschisin function and development of x-linked retinoschisis (see,Wu and Molay, J. Biol. Chem., 278(30):28139-28146, 2003, which isincorporated herein by reference).

In one embodiment, the therapeutic protein comprises at least 50contiguous amino acids, at least 75 contiguous amino acids, at least 125contiguous amino acids, at least 150 contiguous amino acids, at least175 contiguous amino acids or at least 200 contiguous amino acids of ahuman retinoschisin protein, so long as the resulting therapeuticprotein retains at least one function of a full length retinoschisinprotein. In one embodiment, the therapeutic protein comprises at least50 contiguous amino acids, at least 75 contiguous amino acids, at least125 contiguous amino acids, at least 150 contiguous amino acids, atleast 175 contiguous amino acids or at least 200 contiguous amino acidsfrom SEQ ID NO:2, so long as the therapeutic protein retains at leastone function of a full length retinoschisin protein. In one embodiment,the therapeutic protein comprises SEQ ID NO:2 or SEQ ID NO:5. In oneembodiment, the therapeutic protein consists of SEQ ID NO:2 or SEQ IDNO:5.

In one embodiment, a therapeutic protein comprises the discoidin domainof retinoschisin. In one embodiment, a therapeutic protein comprises thediscoidin domain of a human or mouse retinoschisin protein. In oneembodiment, a therapeutic protein comprises the discoidin domain of aprotein comprising SEQ ID NO:2 or SEQ ID NO:5. In one embodiment, atherapeutic protein comprises SEQ ID NO:8.

Therapeutic proteins of the present invention may also be variants ofwild-type proteins. As used herein, a variant refers to a protein, ornucleic acid molecule, the sequence of which is similar, but notidentical to, a reference sequence, wherein the activity of the variantprotein (or the protein encoded by the variant nucleic acid molecule) isnot significantly altered. These variations in sequence can be naturallyoccurring variations, or they can be engineered through the use ofgenetic engineering technique know to those skilled in the art. Examplesof such techniques may be found in Sambrook J, Fritsch E F, Maniatis Tet al., in Molecular Cloning—A Laboratory Manual, 2nd Edition, ColdSpring Harbor Laboratory Press, 1989, pp. 9.31-9.57, or in CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6.

With regard to variants, any type of alteration in the amino acid, ornucleic acid, sequence is permissible so long as the resulting variantprotein retains the function of the wild-type protein. Examples of suchvariations include, but are not limited to, deletions, insertions,substitutions and combinations thereof. For example, with regard toproteins, it is well understood by those skilled in the art that one ormore (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10), amino acids can often beremoved from the amino and/or carboxy terminal ends of a protein withoutsignificantly affecting the activity of that protein. Similarly, one ormore (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids can often beinserted into a protein without significantly affecting the activity ofthe protein.

Any amino acid substitution is permissible so long as the activity ofthe protein is not significantly affected. In this regard, it isappreciated in the art that amino acids can be classified into groupsbased on their physical properties. Examples of such groups include, butare not limited to, charged amino acids, uncharged amino acids, polaruncharged amino acids, and hydrophobic amino acids. Preferred variantsthat contain substitutions are those in which an amino acid issubstituted with an amino acid from the same group. Such substitutionsare referred to as conservative substitutions.

Naturally occurring residues may be divided into classes based on commonside chain properties:

1) hydrophobic: Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr;

3) acidic: Asp, Glu;

4) basic: Asn, Gln, His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve the exchange ofa member of one of these classes for a member from another class.

In making amino acid changes, the hydropathic index of amino acids maybe considered. Each amino acid has been assigned a hydropathic index onthe basis of its hydrophobicity and charge characteristics. Thehydropathic indices are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5). The importance of the hydropathicamino acid index in conferring interactive biological function on aprotein is generally understood in the art (Kyte et al., 1982, J. Mol.Biol. 157:105-31). It is known that certain amino acids may besubstituted for other amino acids having a similar hydropathic index orscore and still retain a similar biological activity. In making changesbased upon the hydropathic index, the substitution of amino acids whosehydropathic indices are within ±2 is preferred, those within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functionally equivalent protein orpeptide thereby created is intended for use in immunological invention,as in the present case. The greatest local average hydrophilicity of aprotein, as governed by the hydrophilicity of its adjacent amino acids,correlates with its immunogenicity and antigenicity, i.e., with abiological property of the protein. The following hydrophilicity valueshave been assigned to these amino acid residues: arginine (+3.0); lysine(+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); and tryptophan (−3.4). In makingchanges based upon similar hydrophilicity values, the substitution ofamino acids whose hydrophilicity values are within ±2 is preferred,those within ±1 are particularly preferred, and those within ±0.5 areeven more particularly preferred. One may also identify epitopes fromprimary amino acid sequences on the basis of hydrophilicity.

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. For example, amino acidsubstitutions can be used to identify important residues of thetherapeutic protein, or to increase or decrease the immunogenicity,solubility or stability of the therapeutic proteins described herein.Exemplary amino acid substitutions are shown below:

Amino Acid Substitutions Original Amino Acid Exemplary Substitutions AlaVal, Leu, Ile Arg Lys, Gln, Asn Asn Gln Asp Glu Cys Ser, Ala Gln Asn GluAsp Gly Pro, Ala His Asn, Gln, Lys, Arg Ile Leu, Val, Met, Ala Leu Ile,Val, Met, Ala Lys Arg, Gln, Asn Met Leu, Phe, Ile Phe Leu, Val, Ile,Ala, Tyr Pro Ala Ser Thr, Ala, Cys Thr Ser Trp Tyr, Phe Tyr Trp, Phe,Thr, Ser Val Ile, Met, Leu, Phe, Ala

As used herein, the phrase “significantly affect a protein's activity”refers to a decrease in the activity of a protein by at least 10%, atleast 20%, at least 30%, at least 40% or at least 50%. Methods ofmeasuring such activities are known to those skilled in the art.

In one embodiment, the therapeutic protein comprises an amino acidsequence at least 95%, at least 98% or at least 99% identical to thesequence of a wild-type retinoschisin protein, so long as the resultingtherapeutic protein retains at least one function of a full lengthretinoschisin protein. In one embodiment, the therapeutic proteincomprises an amino acid sequence at least 95%, at least 98% or at least99% identical to the sequence of a wild-type human, retinoschisinprotein, so long as the resulting therapeutic protein retains at leastone function of a full length retinoschisin protein. In one embodiment,the therapeutic protein comprises an amino acid sequence at least 95%,at least 98% or at least 99% identical to the sequence of SEQ ID NO:(SEQID NO:2 or SEQ ID NO:5), so long as the resulting therapeutic proteinretains at least one function of a full length retinoschisin protein. Inone embodiment, a therapeutic protein comprises an amino acid sequenceat least 90%, at least 95% identical, at least 97% identical to SEQ IDNO:8, wherein the therapeutic protein retains at least one function of afull length retinoschisin protein. In one embodiment, a therapeuticprotein comprises an amino acid sequence at least 90%, at least 95%identical, at least 97% identical to SEQ ID NO:8, wherein the amino acidsequence comprises those cysteine residues necessary for retinoschisinfunction.

A therapeutic molecule may also be a nucleic acid molecule, such as anRNA molecule, that regulates expression of specific genes. For example,a small inhibitory RNA (siRNA) can bind to specific transcripts, therebypreventing such transcripts from being translated. In one embodiment,the therapeutic molecule is a siRNA.

It is well appreciated in the art that the efficiency of delivery ofnucleic acid molecules into cells may be increased using deliveryvehicles such as viral vectors. Thus, in one embodiment, the expressionvector may comprise nucleic acid sequences that allow replication of thevector by viral systems and packaging of the expression vector into aviral vector. One example of such sequences is the inverted terminalrepeat (ITR) sequences found in adeno-associated viruses. Examples ofviral replication systems are known in the art and include, for example,the use of helper viruses (e.g., adenoviruses) as well as recombinantcells expressing proteins that recognize AAV ITR sequences and directreplication of nucleic acid molecules comprising ITR sequences (e.g.,cells expressing AAV Rep proteins). Similarly, packaging systems thatcan package the expression vector into viral vectors are known to thosein the art (e.g., recombinant cells expressing AAV capsid proteins).Thus, in one embodiment, the expression vector comprises at least oneAAV ITR sequence. In one embodiment, the expression vector comprises apair of AAV ITR sequences. AAV ITR sequences useful for constructingexpression vectors of the present invention can be from any AAV so longas they are capable of allowing replication of the expression vector byan AAV replication system, and packaging of the expression vector into aviral vector. In one embodiment, the expression vector comprises atleast one ITR sequence from a virus selected from the group consistingof AAV1, AAV2, AAV4, AAV5, AAV7, AAV8 and AAV9. In one embodiment, theexpression vector comprises at least one ITR sequence from AAV8.

The inventors have found that modification of ITR sequences may resultin an increase in the expression of the therapeutic protein encoded bythe expression vector. For example, it is well-known in the art that ITRsequences contain specific regions, such as the rep nicking sequence andthe D region, that are necessary for proper synthesis of a complementarynucleic acid strand and resolution o the duplex molecule into individualAAV genomes. Removal of one or more of these regions causes failure ofthe duplex genomic nucleic acid molecule to resolve into two individualmolecules, producing in a self-complementary molecule, which results inan increase in expression of the encoded protein. Thus, in oneembodiment, the expression vector comprises at least one ITR that hasbeen modified at the rep nicking sequence or within the D region. In oneembodiment, the expression vector comprises at least one ITR that lacksthe rep nicking sequence. In one embodiment, the expression vectorcomprises at least one ITR that lacks the D region. In one embodiment,the expression vector comprises at least one ITR that lacks the repnicking sequence and the D-region.

As has been discussed, packaging of the expression vector into a viralvector may increase the efficiency of delivery of the expression vectorinto cells of the eye. As used herein, a viral vector refers to aparticle that comprises capsid proteins from one or more viruses, andwhich can encapsulate, or contain, the expression vector within theparticle. Viral vectors may increase the efficiency of delivery bybinding to receptors on the cell surface and becoming internalized(e.g., by fusion with the cell membrane or by endocytosis) therebydelivering the expression vector into the interior of cells of the eye.The capsid proteins of any virus can be used to construct viral vectors,so long as the resulting viral vector is capable of delivering theexpression vector into cells of the eye. Preferred capsid proteins to beused in constructing viral vectors may be obtained from a virus selectedfrom the group consisting of an adeno-associated virus (an AAV virus), acytomegalovirus (CMV), a retrovirus, an adenovirus, a herpes virus, avaccinia virus, a poliovirus, and a Sindbis virus.

In one embodiment, the viral vector comprises capsid proteins from anadeno-associated virus (AAV). AAV is a small (approx. 20 nm indiameter), non-enveloped virus from the parvoviridae family. AAV isdistinct from other members of this family in that it lacks the abilityto replicate by itself and thus relies on the external provision ofreplication and packaging functions. These functions may be supplied bya helper virus or by cells that have been engineered to provide suchfunctions. The genome of AAV virus consists of a single linear segmentthat is approximately 5 kb in length. The ends of the genome consist ofshort inverted repeat (ITR) sequences that fold into T-shaped hairpinstructures that serve as the viral origin of replication. The ITR regioncontains two elements that have been described as central to thefunction of the ITR. These elements are the D region repeat motif andthe terminal resolution site (trs). The repeat motif binds to Repproteins, which are involved in regulation of replication, transcriptionand production of progeny genomes. Binding of the Rep protein positionsthe Rep protein so that it can cleave at the trs.

Currently there are several known AAVs, examples of which include AAV1,AAV2, AAV3, AAV4, AAVS, AAV7, AAV8 and AAV9. The capsid protein from anyAAV can be used so long as the resulting particle is able to encapsulatean expression vector of the present invention and deliver it into cellsof the eye. In a preferred embodiment, the capsid proteins are fromAAV8. Thus, one embodiment of the present invention is a viral vectorcomprising capsid proteins from AAV8 (an AAV8 vector), wherein the viralvector comprises an expression vector of the present invention.

It has been discovered that the presence of human preexisting antibodiesreactive with primate AAV serotypes may reduce the clinical usefulnessof vectors made from these AAV serotypes (Arbetman, et. al., supra). Inparticular, a significant proportion of humans have antibodies thatneutralize AAV serotypes 1 to 6, and experiments have demonstrated thatthe injection of human antibodies into mice to generate sera with lowneutralizing titers significantly reduced transduction with AAV2vectors. To address the problem of human preexisting humoral immunity toAAV serotypes, the viral vectors of the present invention preferablycomprise AAV capsid proteins having little or no preexisting immunity inhumans, including, but not limited to AAV8 capsid proteins.

As used herein, an AAV8 capsid protein refers to a full-length AAV8capsid protein, or any portion thereof that is able to form a viralparticle, encapsulating an expression vector of the preset invention anddelivering the encapsulated expression vector into a cell. In oneembodiment, the viral vector comprises a protein comprising at least 50amino acids, at least 75 amino acids, at least 100 amino acids, at least150 amino acids or at least 200 amino acids from an AAV8 capsid protein.In one embodiment, the viral vector comprises at least 50 amino acids,at least 75 amino acids, at least 100 amino acids, at least 150 aminoacids or at least 200 amino acids from SEQ ID NO:14. In one embodiment,the viral vector comprises a protein comprising SEQ ID NO:14.

Variants of AAV8 capsid proteins can also be used to produce viralvectors of the present invention, so long as the variants protein isable to forming a viral particle, encapsulating an expression vector ofthe preset invention and delivering the encapsulated expression vectorinto a cell. In one embodiment, the viral particle comprises a capsidprotein at least 90% identical, at least 95% identical, at least 97%identical or at least 99% identical to an AAV8 capsid protein. In oneembodiment, the viral particle comprises a capsid protein at least 90%identical, at least 95% identical, at least 97% identical or at least99% identical to SEQ ID NO:14. Methods of the present invention compriseadministering vectors of the present invention to the eye of anindividual in need of such treatment. Any method of administration canbe used to deliver the expression vector, so long as the expressionvector is delivered into the interior of the eye. For example, in oneembodiment the expression vector may be encapsulated in other molecules(e.g., proteins, lipids, etc) such that the encapsulated expressionvector is able to traverse the outer layers of the eye (i.e., cornea,iris, sclera, pupil, lens, or conjunctiva) and enter into theintraocular fluid (also referred to as the aqueous humor). In oneembodiment, the expression vector is encapsulated in a viral vector thatis able to traverse the outer layers of the eye and enter into theintraocular fluid. Thus, in certain embodiments the expression vector isadministered topically to the eye. In preferred embodiments, theexpression vector, either alone or in an encapsulated form, is injectedinto the eye. This may include intramuscular, intradermal, subcutaneous,subconjunctival and sub-Tenon's, intravitreal, subretinal, intravenousand intracameral injections. Such injections can deliver the expressionvector, or a viral vector containing the expression vector, to theintraocular fluid or to a location within the retina. In one embodiment,the injection delivers the expression vector, or a viral vectorcontaining the expression vector, to the intraocular fluid. In oneembodiment, the injection delivers the expression vector, or a viralvector containing the expression vector, into the retina. In oneembodiment, the expression vector is administered by intravitrealinjection. In another embodiment, the expression vector is administeredby subretinal injection. In another embodiment, the expression vector isadministered by sub-Tenon's injection Methods of performing intraocularinjections are known to those skilled in the art. In all of theseembodiments, the expression vector is preferably contained within andadministered via a polypropylene syringe. When administered by thesemeans, the single injection dosage may include between 1e⁸ vg/eye and3e¹³ vg/eye (i.e., 1×10⁸ vector genomes (vg) per eye to 3×10¹³ vectorgenomes per eye). When administered by these means, the single injectiondosage may be between 3e⁸ vg/eye and 1e¹³ vg/eye, or between 1e⁹ vg/eyeand 1e¹³ vg/eye, or between 3e⁹ vg/eye and 1e¹³ vg/eye, or between 1e¹⁰vg/eye and 1e¹³ vg/eye, or between 3e¹⁰ vg/eye and 1e¹³ vg/eye, orbetween 1e¹¹ vg/eye and 1e¹³ vg/eye, or between 3e¹¹ vg/eye and 1e¹³vg/eye, or between 1e¹² vg/eye and 1e¹³ vg/eye, or between 3e¹² vg/eyeand 1e¹³ vg/eye.

The present invention also provides vectors for performing the methodsdisclosed herein. Thus, one embodiment of the present invention is anexpression vector encoding a therapeutic protein for treating a diseaseof the eye, wherein the expression vector expresses high-levels of atherapeutic protein when administered to the eye of an individual inneed of such treatment. In one embodiment, the expression vector is aplasmid. In one embodiment, the expression vector is a linear nucleicacid molecule. In one embodiment, the expression vector comprises DNA.In one embodiment the expression vector comprises RNA. In oneembodiment, the expression vector comprises one or more sequences fromone or more viruses. In a further embodiment, the expression vectorcomprises one or more nucleic acid sequences from an adeno-associatedvirus (an AAV vector), a cytomegalovirus (CMV) (a CMV vector), aretrovirus, an adenovirus, a herpes virus, a vaccinia virus (a vacciniavector), a poliovirus, a Sindbis virus, or any other DNA or RNA virus.In one embodiment, the expression vector comprises nucleic acidsequences from an AAV selected from the group consisting of AAV1, AAV2,AAV4, AAV5, AAV7, AAV8 and AAV9. In a preferred embodiment, theexpression vector comprises nucleic acid sequences from AAV8.

Expression vectors of the present invention provide high-levelexpression of therapeutic molecules capable of alleviating the symptomsof a disease of the eye. Thus, one embodiment of the present inventionis an expression vector encoding a therapeutic molecule, wherein theexpression vector comprises a promoter that drives high-level expressionthe therapeutic molecule. In one embodiment, the promoter is aneye-specific promoter. In one embodiment, the promoter is aretina-specific promoter. In one embodiment, the promoter comprises atleast a portion of a retinoschisin promoter. In one embodiment, theportion is from SEQ ID NO:9. In one embodiment, the promoter comprisesSEQ ID NO:9. In one embodiment, the promoter consists of SEQ ID NO:9. Inone embodiment, the promoter comprises a nucleotide sequence at least95% identical to at least one sequence selected from the groupconsisting of SEQ ID NO:10 and SEQ ID NO:11, wherein the promoter hasretinoschisin gene promoter activity. In one embodiment, the promotercomprises at least one sequence selected from the group consisting ofSEQ ID NO:10 and SEQ ID NO:11, wherein the promoter has retinoschisingene promoter activity. In one embodiment, the promoter comprises SEQ IDNO:9.

Expression vectors of the present invention may also comprise promotersthat have been modified in order to increase or decrease the expressionof the encoded therapeutic molecule. Thus, in one embodiment, theexpression vector comprises a promoter, such as a retinoschisin promoterthat has been modified by mutation of the promoter sequence. In oneembodiment, the expression vector comprises a promoter that is lackingone or more genetic element, such as, a TATA element, a B recognitionelement or an enhancer element. One embodiment is an expression vectorencoding a therapeutic molecule, wherein the encoding sequence is linkedto a promoter that drives high level expression of the encoded molecule,wherein the expression vector comprises an enhancer element. In oneembodiment, the enhancer element is an IRBP enhancer element. In oneembodiment, the enhancer element comprises SEQ ID NO:12. In oneembodiment, the enhancer element comprises a nucleotide sequence atleast 95% identical to SEQ ID NO:12, wherein the enhancer retains theability to enhance transcription from a nearby promoter (i.e., apromoter within 500 nucleotides of either end of the enhancer sequence).In one embodiment, the IRBP enhancer element is linked to one end of theeye-specific promoter. In one embodiment, the IRBP enhancer element isinserted within the sequence of the eye-specific promoter. In oneembodiment, the IRBP enhancer element is inserted within the sequence ofthe retinoschisin gene promoter.

As noted, expression vectors of the present invention encode therapeuticmolecules for the treatment of diseases of the eye. The therapeuticmolecule can be any molecule that is useful for treating disease of theeye when expressed in cells of the eye. Thus, one embodiment of thepresent invention is an expression vector encoding a therapeuticmolecule, wherein the expression vector comprises a promoter that driveshigh-level expression of the therapeutic molecule in the eye, andwherein the expression of the therapeutic molecule in cells of the eyealleviates the symptoms of a disease of the eye. In one embodiment, thetherapeutic molecule is an RNA molecule. In one embodiment, thetherapeutic molecule is an siRNA molecule. In one embodiment, thetherapeutic molecule is a protein. In one embodiment, the therapeuticmolecule is a protein normally found in the eye. In one embodiment, thetherapeutic molecule is a protein comprising at least a portion of aretinoschisin protein, wherein the encoded protein has retinoschisinprotein activity. In one embodiment, the therapeutic molecule is aprotein that comprises at least 50 amino acids, at least 75 amino acids,at least 100 amino acids, at least 150 amino acids, or at least 200amino acids from a retinoschisin protein, wherein the encoded proteinhas retinoschisin protein activity. In one embodiment, the therapeuticmolecule is a protein that comprises at amino acids 63-224 from aretinoschisin protein, wherein the encoded protein has retinoschisinprotein activity. In one embodiment, the therapeutic molecule is aprotein that comprises at least 50 amino acids, at least 75 amino acids,at least 100 amino acids, at least 150 amino acids, or at least 200amino acids from SEQ ID NO:2. In one embodiment, the therapeutic proteincomprises at least amino acids 63-224 from SEQ ID NO:2, wherein theencoded protein has retinoschisin activity. In one embodiment, thetherapeutic protein comprises SEQ ID NO:2 or SEQ ID NO:5. In oneembodiment, the therapeutic protein consists of SEQ ID NO:2 or SEQ IDNO:5.

Therapeutic proteins encoded by expression vectors of the presentinvention may also be variants of proteins that alleviate the symptomsof a disease of the eye. Such variants may comprise one or more aminoacid substitutions, deletions or insertions. Thus, one embodiment of thepresent invention is an expression vector encoding a variant of awild-type therapeutic protein, wherein the expression vector comprises apromoter that drives high-level expression the variant protein in theeye, and wherein the expression of the variant protein in cells of theeye alleviates the symptoms of a disease of the eye. In one embodiment,the therapeutic protein comprises an amino acid sequence at least 95%,at least 98% or at least 99% identical to the sequence of a wild-typetherapeutic protein, wherein the variant protein retains the function ofthe wild-type protein. In one embodiment, the expression vector encodesa variant of a wild-type retinoschisin protein. In one embodiment, theencoded protein comprises an amino acid sequence at least 95%, at least98% or at least 99% identical to the sequence of a wild-typeretinoschisin protein, wherein the encoded protein has retinoschisinactivity. In one embodiment, the encoded protein comprises an amino acidsequence at least 95%, at least 98% or at least 99% identical to thesequence of a wild-type human, retinoschisin protein, wherein theencoded protein has retinoschisin activity. In one embodiment, thetherapeutic protein comprises an amino acid sequence at least 95%, atleast 98% or at least 99% identical to the sequence of SEQ ID NO:(SEQ IDNO:2 or SEQ ID NO:5), wherein the encoded protein has retinoschisinactivity.

Expression vectors of the present invention may be packaged into viralvectors in order to improve the efficiency of their delivery. Thus, oneembodiment of the present invention is an expression vector encoding atherapeutic molecule, wherein the expression vector comprises a promoterthat drives high-level expression the therapeutic molecule in the eye,wherein the expression of the therapeutic molecule in cells of the eyealleviates the symptoms of a disease of the eye, and wherein the vectorcomprises nucleic acid sequences that direct the replication of,transcription from, or packaging of the expression vector. In oneembodiment, the expression vector comprises a sequence that allows thepackaging of the expression vector into a viral vector. In oneembodiment, the expression vector comprise one or more ITR sequencesfrom a virus selected from the group consisting of AAV1, AAV2, AV4,AAV5, AAV7, AAV8 and AAV9. In one embodiment, the expression vectorcomprises one or more ITR from AAV8. In one embodiment, the expressionvector comprises at least one ITR sequence comprising at least a portionof an AAV8 ITR, wherein the ITR is still able to direct replication,transcription and packaging of the expression vector.

ITRs of expression vectors of the present invention may also be modifiedto improve the characteristics of the expression vector. Thus, oneembodiment of the present invention is an expression vector encoding atherapeutic molecule, wherein the expression vector comprises a promoterthat drives high-level expression the therapeutic molecule in the eye,wherein expression of the therapeutic molecule in cells of the eyealleviates the symptoms of a disease of the eye, and wherein the vectorcomprises two or more AAV ITR sequences, wherein at least one ITR lacksthe rep nicking sequence or the D region. In one embodiment, theexpression vector comprises at least one ITR that lacks a rep nickingsequence. In one embodiment, the expression vector comprises at leastone ITR that lacks a D region sequence. In one embodiment, theexpression vector comprises at least one ITR that lacks both the repnicking sequence and the D region sequence.

The present invention also provides viral vectors useful for practicingthe disclosed methods. Viral vectors of the present invention comprisean expression vector of the present invention encapsulated in viralcapsid proteins. Encapsulation of expression vectors within such capsidproteins increases the efficiency with which expression vectors aredelivered into cells of the eye. Viral vectors of the present inventionproduce either insignificant, or no, immune response when administeredto the eye of an individual. Without being bound by theory, theinventors believe that this is because the level of expression of thetherapeutic protein is high enough that the dose of viral vectorrequired to alleviate symptoms of the disease being treated may be lowenough that it either fails to elicit an immune response or it elicitsan insignificant immune response. In this context, an “insignificantimmune response” means an immune response that this either not therapylimiting, or is not dose limiting or may be clinically managed byadjusting the dosage amount or timing or by the concurrentadministration of anti-inflammatory agent (steroidal or non-steroidal),or a combination of these factors. Thus, one embodiment of the presentinvention is a viral vector comprising an expression vector encoding atherapeutic protein for treating a disease of the eye, wherein theexpression vector expresses high-levels of a therapeutic protein whenadministered to the eye of an individual, wherein the expression vectoris encapsulated by capsid proteins from one or more viruses. In oneembodiment, the one or more viruses are selected from the groupconsisting of an adeno-associated virus (an AAV virus), acytomegalovirus (CMV), a retrovirus, an adenovirus, a herpes virus, avaccinia virus, a poliovirus, and a Sindbis virus. In a preferredembodiment, the viral capsid proteins are from AAV. In one embodimentthe viral capsid proteins are from AAV8. In one embodiment, the viralcapsid protein comprises SEQ ID NO:14.

Viral vectors of the present invention may also be constructed usingfunctional portions of viral capsid proteins. Thus, one embodiment ofthe present invention is a viral vector comprising an expression vectorencoding a therapeutic molecule for treating a disease of the eye,wherein the expression vector expresses high-levels of a therapeuticmolecule when administered to the eye of an individual, and wherein theexpression vector is encapsulated by proteins comprising at least aportion of a capsid protein from one or more viruses, wherein theproteins comprising at least a portion of a capsid proteins from one ormore viruses self assemble into a viral vector. In one embodiment, theencapsulating proteins comprise at least a portion of capsid proteinfrom one or more AAVs. In one embodiment, the encapsulating proteinscomprise at least 50 amino acids, at least 75 amino acids, at least 100amino acids, at least 150 amino acids or at least 200 amino acids from acapsid protein from one or more AAVs. In a preferred embodiment, theencapsulating protein comprises at least a portion of an AAV8 capsidprotein. In one embodiment, the encapsulating proteins comprise at least50 amino acids, at least 75 amino acids, at least 100 amino acids, atleast 150 amino acids or at least 200 amino acids from an AAV8 capsidprotein. In one embodiment, the encapsulating protein comprises at least50 amino acids, at least 75 amino acids, at least 100 amino acids, atleast 150 amino acids or at least 200 amino acids from SEQ ID NO:14.

Viral vectors of the present invention may also be constructed usingvariants of viral capsid proteins. Thus, one embodiment of the presentinvention is a viral vector comprising an expression vector encoding atherapeutic molecule for treating a disease of the eye, wherein theexpression vector expresses high-levels of a therapeutic molecule whenadministered to the eye of an individual, and wherein the expressionvector is encapsulated by proteins comprising an amino acid sequence atleast 90% identical, at least 95% identical, at least 97% identical orat least 99% identical to a capsid protein from a virus selected from anadeno-associated virus (an AAV virus), a cytomegalovirus (CMV), aretrovirus, an adenovirus, a herpes virus, a vaccinia virus, apoliovirus, and a Sindbis virus, wherein the encapsulating proteins areable to self-assemble into a viral vector. In one embodiment, theencapsulating proteins comprise an amino acid sequence at least 90%identical, at least 95% identical, at least 97% identical or at least99% identical to an AAV capsid protein. In a preferred embodiment, theencapsulating proteins comprise an amino acid sequence at least 90%identical, at least 95% identical, at least 97% identical or at least99% identical to an AAV8 capsid protein. In one embodiment, theencapsulating proteins comprise an amino acid sequence at least 90%identical, at least 95% identical, at least 97% identical or at least99% identical to SEQ ID NO:14.

The present invention also provides methods for producing viral vectorsfor use in methods of the present invention. Thus, one embodiment of thepresent invention is a method to produce a viral vector for treating adisease of the eye, comprising contacting an expression vector of thepresent invention with a packaging system, wherein the expression vectorcomprises sequences that direct packaging of the expression vector intoa viral vector. In one embodiment, the expression vector encodes atherapeutic molecule for treating a disease of the eye, wherein theexpression vector expresses high-levels of the therapeutic molecule whenadministered to the eye of an individual, and wherein the expressionvector comprises nucleic acid sequences that direct packaging of theexpression vector. In one embodiment, the expression vector comprisesone or more AAV ITRs. In one embodiment, the step of contacting theexpression vector with a packaging system comprises introducing theexpression vector into a cell. Methods of introducing nucleic acidmolecules into cells are known in the art and include, for example,transfection and electroporation. In one embodiment, the expressionvector is introduced into a cell expressing one or more AAV proteins. Inone embodiment, the cell expressing one or more AAV proteins is arecombinant protein engineered to express an AAV Rep protein, an AAVcapsid protein, or both the Rep protein and a capsid protein. Packagingfunctions may also be provided by a helper virus. Thus, in oneembodiment, the expressing vector is introduced into a cell that is alsoinfected with a helper virus.

The present invention also provides therapeutic compositions comprisingexpression vectors of the present invention. Such compositions compriseexpression vectors in physiologically acceptable solutions thatcomprise, for example, water, saline, salts, buffer, diluents,stabilizing agents, polymers, chelating agents and the like. One exampleof a physiologically acceptable solution is a solution comprising about10 mM Tris-HCl (pH 7.4) and about 180 mM NaCl. A further example of asuitable solution is a solution that comprises about 310 mM Tris-HCl (pH7.4), about 180 mM NaCl, and about 0.001% Pluronic F-68. In a preferredembodiment, a composition of the present invention comprises a solutioncomprising about 10 mM NaPhosphate (pH 7.3), about 180 mM NaCl, andabout 0.001% Pluronic F-68. It will be appreciated by those skilled inthe art that such concentrations are approximate and may vary by as muchas 10% or more, without significant affect on the efficacy of thecomposition.

The present invention also provides kits for practicing the disclosedmethods. Kits of the present invention may comprise expression vectorsof the present invention and viral vectors of the present invention.Such kits may also comprise reagents and tools necessary for practicingthe disclosed methods such as, for example, buffers, diluents, syringes,needles and instructions for administering such reagents. While thepresent invention has been described with reference to the specificembodiments thereof, it should be understood by those skilled in the artthat various changes may be made and equivalents may be substitutedwithout departing from the true spirit and scope of the invention. Inaddition, many modifications may be made to adapt a particularsituation, material, composition of matter, process, process step orsteps, to the objective, spirit and scope of the present invention. Allsuch modifications are intended to be within the scope of the claims.

EXAMPLES

These Examples demonstrates the ability of an AAV vector expressing thehuman retinoschisin protein (SEQ ID NO:2) to preserve retinal functionin a mouse model of retinoschisis.

Example 1

A study was conducted to evaluate the ability of the proposed clinicaladeno-associated virus (AAV) retinoschisin vector, AAV8 scRS/IRBP hRS,to preserve retinal function and structure, and to mediate retinoschisinprotein expression when administered intravitreally to the retinoschisindeficient Rs1-KO mouse. AAV8 scRS/IRBP hRS vector at doses of 1.0e6,1.0e7, 5.0e7, 1.0e8, 5.0e8 and 2.5e9 vg/eye, or vehicle, wereadministered by intravitreal injection to 18-34 day old Rs1-KO mice. Thecontralateral eye was not injected. Corneal electroretinogram (ERG)a-wave and b-wave amplitudes were measured at 11 to 15 weeks and 6 to 9months post injection (PI), retinal cavity formation was measured byoptical coherence tomography (OCT) at 12 to 16 weeks PI, andretinoschisin protein expression was measured by immunohistochemistry at12 to 18 weeks and 6 to 9 months PI. ERG and OCT are used clinically asindicators of retinal function and structure, respectively. At 11-15weeks PI, eyes receiving doses of 5e⁷, 1e⁸ and 2.5e⁹ vg/eye showedstatistically-significant improvement in ERG a-wave amplitudes, anddoses of 5e⁷, 1e⁸, 5e⁸, and 2.5e⁹ vg/eye showed statisticallysignificant improvement in ERG b-wave amplitudes compared to uninjectedeyes. Three vector doses were tested at the 6-9 month time point, 1e⁸,5e⁸ and 2.5e⁹ vg/eye, and all produced statistically significantimprovement in ERG a- and b-wave amplitudes compared to uninjected eyes.Retinal cavities, as measured by OCT at 11-15 weeks PI, were alsosignificantly reduced at doses of 5e⁷, 1e⁸, 5e8 and 2.5e⁹ vg/eyecompared to untreated eyes. Retinal immunohistochemistry indicated thatsignificant retinoschisin protein levels were produced at doses of 1e⁷,1e⁸, 5e⁸, and 2.5e⁹ vg/eye, compared to untreated eye at 11-15 weeks PI.Doses of 1e⁸, 5e⁸ and 2.5e⁹ produced expression at 25% of wild type miceor greater. At 6-9 months PI, only the 2.5e⁹ vg/eye dose was tested, andit produced 65% of the wild type retinoschisin level, a significantincrease over 11-15 weeks. These data demonstrate that AAV8 scRS/IRBPhRS shows efficacy at 10- to 100-fold lower doses than the previouslypresented vector AAV8 hRSp4. AAV8 scRS/IRBP hRS was developed byexamination of several retinoschisin vectors for efficacy in the mouseretinoschisis model, and for toxicity in rabbits.

As noted above, this study examined the ability of AAV8 scRS/IRBP hRSvector to preserve retinal function and retinal structure and to mediateretinoschisin protein expression when administered intravitreally to theretinoschisin deficient Rs1-KO mouse. The Rs1-KO mouse is aretinoschisin knockout model of X-linked retinoschisis, that exhibitsstructural and functional changes characteristic of the human diseaseincluding a much reduced b-wave relative to the a-wave, and the presenceof schisis or splitting of the inner nuclear and outer plexiform layers.In this study, vehicle or AAV8 scRS/IRBP hRS vector at doses of 1.0e6,1.0e7, 5.0e7, 1.0e8, 5.0e8 and 2.5e9 vg/eye were administered byintravitreal injection to 18-34 day old Rs1-KO mice. Although a verycrude estimation, mice of this age correspond roughly to the age of thepatient population that might benefit most from a successful therapy(adolescents and young adults). The mice were then evaluated by ERG forretinal function, OCT for retinal structure and immunohistochemistry forretinoschisin expression at 11-15 weeks and 6-9 months PI.

The experiments disclosed herein were also designed to determine thedosage range over which this vector significantly preserves retinalfunction and structure in the Rs1-KO mouse and achieves significantretinal expression of protein.

Description of the Viral Vector Used in this Example

The vector used in this Example, AAV8 scRS/IRBP hRS, is anadeno-associated virus type 8 vector that delivers a self-complementaryvector genome composed of a modified human retinoschisin promoter thatdrives the expression of a human retinoschisin cDNA. This vector alsoemploys an interphotoreceptor retinoid-binding protein (IRBP) enhancerto augment promoter activity, a truncated retinoschisin first intron,and a human beta-globin 3′ untranslated region and polyadenylation site.The structure of AAV8 scRS/IRBP hRS is shown in FIG. 1 and the completesequence of the vector is provided as SEQ ID NO: 16.

Structure of pAAV scRS/IRBP hRS Vector Production Plasmid

The production plasmid for the AAV8 scRS/IRBP hRS vector, called pAAVscRS/IRBP hRS, is composed of the human retinoschisin expressioncassette described above, bounded by AAV2 inverted terminal repeatsequences, that has been cloned into a pBluescript plasmid (StratageneInc., San Diego, Calif.).

Construction of the pAAV scRS/IRBP hRS Vector Production Plasmid

Retinoschisin expression cassette: The protein coding portion of theexpression cassette is composed of a human retinoschisin cDNA thatretains a 319 bp, truncated retinoschisin first intron. The truncatedintron consists of base pairs +95 to +355 and +14396 to +14445 relativeto the retinoschisin transcriptional start site. These sequences encodethe splice donor and lariate/splice acceptor elements, respectively. An8-base pair AsiSI restriction site was introduced between the two partsof the intron to facilitate vector construction. Transcription of theexpression cassette is driven by the human retinoschisin genomicsequence that extends from position −739 relative to the transcriptionstart site, to position+42, which is the base that precedes the startcodon. This promoter sequence contains a 308 bp Alu repeat sequence atpositions −496 to −188, which has been deleted and replaced with a 261bp enhancer from the human IRBP gene, which was flanked by SalI sites.The IRBP enhancer is located at position −1374 to −1635 relative to theIRBP transcriptional start site. Polyadenylation is directed by a 218 bpfragment encoding the entire human beta-globin 3′ untranslated regionand polyadenylation site. This region corresponds to the 218 bp genomicsequence that directly follows the human beta-globin stop codon.Synthetic DNA encoding XhoI and BglII sites was introduced between theretinoschisin stop codon, and the beta globin sequences that encode the3′ untranslated and polyadenylation site, to facilitate construction. ANotI site and an AscI site were added to the 5′ and 3′ ends of theexpression cassette, respectively, in order to ligate it to the 5′ and3′ AAV2 inverted terminal repeat elements.

AAV inverted terminal repeat sequences: The ITRs used in this constructare not identical. The 5′ ITR was derived from psub201. ITRs derivedfrom psub201 have a 15-base pair deletion of the ITR sequence in the Aregion of the palindrome which is not proximal to the transgene.Consequently, they are 130 bps in length rather than the wild typelength of 145 bp. The 5′ ITR was further modified by removal of the “Dregion” which contains the rep nicking site. To do this, the MscI sitelocated near the inside border of the ITR palindrome was cleaved withMscI and a poly linker encoding SmaI (half site)-BamHI-SpeI-XbaI-NotIwas ligated to it, in place of the D region. This modified ITR allowsproduction of self-complementary AAV vector genomes. A PacI site wasalso added to the outside (not proximal to transgene) of the ITR. The 3′ITR is a full length, 145 bp ITR and was produced by nucleic acidsynthesis (Blue Heron, Bothell, Wash.). It is flanked in the inside(proximal to transgene) by an AscI site and an FseI site on the outside.The ITRs are linked to the expression cassette through the NotI (5′) andAsiSI (3′) sites and to the pBluescript plasmid backbone through Pact(5′) and FseI (3′) sites. The promoter of the expression cassette isproximal to the 5′ ITR.

pBluescript plasmid backbone: The pBluescript S/K+plasmid was modifiedfor use as an AAV vector plasmid backbone. The sequence between theAfllII and BstUI (partial) sites located at positions 457 and 1150 inthe plasmid were removed and replaced with synthetic DNA encoding therestriction sites SseI-PvuII-SseI-AflII. This poly linker was furthermodified by ligating a poly linker encoding restriction sitesSseI-PspXI-PmlI-PvuII-SseI between the two SseI sites of the firstpolylinker. Finally, the ITR-flanked AAV vector was excised from thepUC18 used for its construction using by cutting with PspXI and SapI(blunt) and was ligated between the PspXI and PvuII sites of thepolylinker above to create the pAAV scRS/IRBP hRS production plasmid.

Construction of the Viral Vector

The AAV8 scRS/IRBP hRS vectors were prepared as previously described(Grimm D et al. 2003). Briefly, 293 cells cultured in 850 cm² rollerbottles in DME (High Glucose) media containing 10% fetal bovine serum(HyClone SH30070.03) and supplemented with penicillin, streptomycin andglutamine were transiently transfected with the helper plasmids pLadeno5(encoding adenovirus type 2 E2A, E4, and VA RNAs) and pHLP19-8 (encodingAAV2 rep and AAV8cap), and the pAAV scRS/IRBP hRS vector plasmid usingthe calcium phosphate method. After transfection, the media was changedand replaced with the same media lacking serum. Sixty hours later, thecells were collected by centrifugation and stored at −80 C. To purifythe viral vectors, the cell pellets were thawed, suspended in 50 mMTris-HCl, 150 mM NaCl, 2 mM MgCl₂, pH 8.0 and disrupted by 3 rounds ofmicrofluidization. The cell debris was removed by centrifugation and thesupernatant was adjusted to 25 mM CaCl₂ and the resulting pellet wasalso removed by centrifugation. Benzonase nuclease was added to thesupernatant to a final concentration of 100 units per ml and the mixturewas incubated for 1 hour at 37 C. 40% polyethylene glycol 8000 (PEG)/2.5M NaCl was then added to produce final concentrations of 8% PEG and0.650 M NaCl and the vector fraction was precipitated and collected bycentrifugation. The vector fraction was solubilized in 50 mM HEPES, 150mM NaCl, 20 mM EDTA, 1% sodium lauroyl sarcosinate, 10 μg/ml RNase A,pH8.0. This solution applied to a CsCl step gradient and vector wasseparated from the bulk protein and nucleic acids byultracentrifugation. The vector fraction was collected and applied to alinear CsCl gradient and repurified. The purified vector fraction wascollected, dialyzed against 10 mM Tris-Cl, 180 mM NaCl pH 7.4,formulated in 310 mM Tris-Cl, 180 mM NaCl, 0.001% Pluronic F-68 pH 7.4,filter sterilized, and stored at −80 C.

Analysis of the Purified pAAV scRS/IRBP hRS Vector

The purified vector particles were analyzed by Q-PCR Protein assay(BCA), SDS PAGE, Endotoxin assay (type) and Dynamic Light Scattering.Q-PRC assay was performed using the Taqman Real-Time PCR assay withupstream and downstream primers located in retinoschisin exons 3 and 4,respectively, and a probe that spanned the exon 3/4 junction. The levelof protein present was determined by the Bradford method using bovinegamma-globulin as the standard (Rio-Rad, Richmond, Calif.). Thetheoretical protein concentration was calculated as the mass of AAVcapsid protein/ml, based on the Q-PCR result. SDS PAGE analysis wasconducted on 1.2e10 vg (prep 1) and 2e10 vg (prep 2) using 7.5% SDSgels. Visualizataion of the separated proteins was done by staining thegels with Coomassie 8250 or a silver staining. The Kinetic LAL endotoxinassay was performed using a Kinetic Chromogenic Limulus AmoebocyteLyasate Endotoxin Assay Kit (Clonegen Laboratories). The Dynamic LightScattering Assay was performed using a Viscotec 802 DLS instrument, withvector concentrations of 2.25e12 vg/ml and 5e12vg/ml for the AAV8 hRSp4and AAV8 hRS/IRBP vectors, respectively. The results of this analysisare shown in FIG. 2 and tabulated as follows:

Analysis Results for AAV8 scRS/IRBP hRS Vector

Prep 1 Prep 2 Vehicle Vector genomes/ml  2¹²   2¹² Protein/ml (ug/ml)Not Detectable 26 Theoretical 18 18 protein/ml Endotoxin units/ml Notdetectable 12 0.174 SDS PAGE Prominent capsid band at 25 kd. Two minorbands below Staining also above 25 kd and in well Dynamic Light Majorsymmetrical peak at 15-16 nm Scattering for both; No other significantpeaks

Dosage Preparation and Administration

Under a dissecting microscope, one microliter (μl) of vector or vehiclewas administered to the right or left eye by intravitreal injectionusing 10 μl Nanofil syringes (World Precision Instruments, Inc.,Sarasota, Fla.) and removable 35 gauge needle. Injected material wassterilized by passage through a 0.22 μl filter, and the syringes wereloaded under aseptic conditions. Mice were anesthetized with IPketamine, 80 mg/kg, and xylazine, 4 mg/kg, and one drop of 0.5%tetracaine was applied topically on the cornea. One microliter of vectoror vehicle solution was injected through the pars plana in the superiornasal quadrant approximately 1 mm posterior to the limbus in one eye ofeach mouse. The injection volume is approximately one-fifth of the totalvitreous volume. The injection was performed such that the needle tipwas positioned in the center of the vitreous before the vector wasdelivered at a slow rate. The needle was then carefully extracted fromthe eye, and triple antibiotic ophthalmic ointment (neomycin, polymixinB and bacitracin) was applied to the injection site. The mice wereplaced onto a warming plate (35 C to 37 C) until they recovered from theanesthesia and were then put back into their cage.

Test System

The retinoschisin knockout (Rs1-KO) mouse model was generated in 2003.Since November 2003, these mice have been housed at NIH in a sharedanimal facility maintained by the National Institute of Allergy andInfectious Diseases (NIAID) and backcrossed more than 18 generationsonto the C57BL/6J line (Jackson Laboratory, Bar Harbor, Me.). The Rs1-KOmice were 18 to 34 days at injection and 14 to 37 weeks at ERG, andconfirmed to lack retinoschisin expression in the retina and have aretinal structural and functional phenotype similar to that of XLRSpatients, including a reduced b-wave amplitude relative to the a-waveamplitude, and the presence of “schisis” cavities involving splitting orseparations within the outer plexiform (OPL) and inner nuclear layers(INL).

Dosing: 28 mice received 2.5e9 vg/eye. 41 mice received 5.0e8 vg/eye. 39mice received 1.0e8 vg/eye. 26 mice received 5.0e7 vg/eye. 26 micereceived 1.0e7 vg/eye. 26 mice received 1.0e6 vg/eye. 43 mice receivedinjection vehicle.

The study included 229 male Rs1-KO mice. The retinoschisin vector wasadministered unilaterally to 186 Rs1-KO, and the contralateral eye wasnot injected. The mice came from 60 litters which were the offspring of26 different homozygous and 6 heterozygous female Rs1-KO mice crossedwith C57BL/6J males. Vehicle was administered unilaterally to 43 maleRs1-KO mice, and the contralateral eye was not injected. These mice camefrom 15 litters that were the offspring of 13 different homozygousfemale Rs1-KO mice crossed with C57BL/6J male mice. As homozygous Rs1-KOfemales produce only Rs1-KO males it was not necessary to check thegenotype of these males, but several males from this mating scheme wererandomly picked to confirm the genotype. The genotypes of allheterozygous females and their offspring were confirmed by genotyping.

Experimental Procedure

A listing of all mice used in this study, their parents, the testmaterial they received, and their dates of birth, injection, ERG, OCT,and histological examination/sacrifice was compiled and recorded.

Injections

One microliter of AAV8 RS/IRBP hRS vector solution was administeredunilaterally by intravitreal injection on postnatal day (p) 18-25 (21±2days, mean±1 SD) to Rs-1KO mice under aseptic conditions. Control Rs-1KOmice received unilateral, intravitreal injections of 1 μl of vehicle at18-34 PND (24±5 days). One microliter of AAV8 hRS1/IRBP or vehicle wereadministered to 229 Rs1-KO mice: 2.5e9 vg/eye, 28 mice; 5.0e8 vg/eye, 41mice; 1.0e8 vg/eye, 39 mice; 5.0e7 vg/eye, 26 mice; 1.0e7 vg/eye, 26mice; 1.0e6 vg/eye, 26 mice; vehicle, 43 mice. Ocular changes, such ascorneal opacity, and the amount of reflux from the injection site, werenoted for each animal during or immediately following injection. Theinjections of all 186 vector injected animals were successful, but 2mice (1.1%) died after injection while still anesthetized.

Three vector injected mice had to be euthanized before the ERG wascompleted: 2 mice were sacrificed within 3 days of injection becausethey were thought too small to survive; 1 mouse (0.5%) had to besacrificed before ERG recording due to malocclusion, a problem withexcessive tooth growth that occurs in 0.05-0.09% of C57BL mice dependingon substrain. One vehicle injected animal (2.3%) was sacrifice beforethe ERG because of malocclusion and one animal was sacrificed because ofan occluded eye. Twenty-five vector injected animals (13%) died duringor after anesthesia for ERG or OCT. Two vehicle injected animals (4.6%)died after the ERG was complete.

The injection of vector was not considered relevant to the deaths ofanimals prior to anesthesia for the ERG: 2 mice that died beforerecovering from injection anesthesia, 2 mice sacrificed immediately forsmall size, and one mouse sacrificed for malaocclusion. Overall, 25animals in the vector injected group died during or after anesthesia forthe ERG or OCT, but the number of deaths is not statistically greaterthan in the vehicle injected control group (P=0.18, Fisher's exact test)and did not show a significant trend with dose (P=0.72, Chi-square testfor trend).

One-third of the deaths in the vector injected group occurred in thecohort receiving the dose of 1e6 vg/eye, and when each dose was comparedseparately to vehicle, only this dose was statistically greater thanvehicle (P=0.005). As this was the lowest dose, these deaths may reflectpreexisting condition of the animals or technical manipulation ratherthe effect of vector.

Electroretinogram (ERG)

ERG Recording Procedure:

To evaluate the efficacy of the retinoschisin vector AAV8 scRS/IRBP hRSin preserving retinal function in Rs1-KO mice, the dark adaptedelectroretinogram (ERG) was recorded in both eyes simultaneously between11 and 15 weeks after intravitreal injection of vector (“short term”),and/or between 6 and 9 months after intravitreal injection of vector(“long term”). Vehicle control mice were recorded between 14 and 18weeks after intravitreal injection.

The day before recordings, mice were moved from the animal facility tothe lab for overnight dark adaption in a light tight ventilated box. Allsubsequent procedures were performed in dim red light or darkness. Afteranesthesia with 80 mg/kg ketamine and 4 mg/kg xylazine given byintraperitoneal injection, the pupils were dilated with topical 0.5%tropicamide and 0.5% phenylephrine HCL, and the mouse was placed on aheating pad at 37° C. One percent proparacaine topical anesthesia wasput on the cornea before placing gold wire loop active and referenceelectrodes in the center of the cornea and on the edge of the sclera,respectively. Recordings were the average of 1 to 20 dark-adaptedresponses to 10 μs flashes presented in 0.5 log unit intensity stepsfrom −6.9 to +0.6 log cd·s/m² in a Ganzfeld (full field) bowl. Theintensities to elicit these responses in mouse are similar to those inhuman. The main difference is that only about 3% of mouse photoreceptorsare cones, compared to 5% for humans, therefore the relativecontributions from cone photoreceptors is much less at the maximumintensity eliciting a mixed rod-cone response. Total time for eachrecording was about 20 minutes, and following recording, mice wereallowed to recover on a heating pad before being replaced in cages.

The ERG signals were amplified 5000 times and filtered by a 0.1 to 1 kHz3 db/decade bandpass and a 60 Hz line filter using a Grass CP511 ACamplifier before being digitized with a National Instruments AD board at5 kHz. One to 20 waveforms were collected and averaged at eachintensity, with smaller numbers collected at higher intensities.

ERG Data Analysis:

ERG results reported in this study are a-wave and b-wave amplitude inresponse to a single stimulus intensity of 0.6 log cd·s/m². Thedark-adapted a-wave reflects the activation phase of rod photoreceptorsin response to light; the b-wave results from the response of bipolarcells which are activated transynaptically by photoreceptors. Thevehicle group was used to control for possible effects of the injectionprocedure and was analyzed at the short term time point when maximumtreatment effect of vector was expected.

Statistical Procedures:

Treated a-wave and b-wave amplitudes in vector and vehicle injected eyeswere compared to a-wave and b-wave amplitudes in the untreated eyes atthe short term time point and in the three highest dose vector injectedgroups at the long term time point using unpaired t tests corrected formultiple comparisons with the Holm-Sidak method assuming populationswith the same standard deviation. All statistics were performed usingGraphpad Prism 6.0. (GraphPad Prism version 6.00 for Windows).

Ocular Coherence Tomography (OCT)

The retinas of both eyes of all mice that received vector and survivedthe short term ERG and had unaltered ocular media were imaged in vivo byOCT from 2 to 21 days after the ERG. The numbers of mice imaged in eachdose group include: 2.5e9 vg/eye, 25 Rs1-KO mice imaged; 5.0e8 vg/eye,25 Rs1-KO mice imaged; 1.0e8 vg/eye, 20 Rs1-KO mice imaged; 5.0e7vg/eye, 19 Rs1-KO mice imaged; 1.0e7 vg/eye, 25 Rs1-KO mice imaged;1.0e6 vg/eye, 16 Rs1-KO mice imaged.

The OCT imaging system acquires, processes, displays and savesdepth-resolved images of retinal tissue microstructure in vivo. We usedthe ultra-high resolution spectral domain OCT from Bioptigen, whichallows noninvasive non-contact imaging providing microscopic tomographicimages of the retina with 2 micron axial resolution. OCT imaging in XLRSpatients has been demonstrated to be a useful tool in conjunction withfunctional measures to characterize retinal pathology. Mice wereanesthetized (80 mg/kg ketamine and 4 mg/kg xylazine), mounted in acustom holder, and the optic nerve head of the retina was placed at thecenter of a rectangular scan area of 1.4 mm×1.4 mm (0.7 mm on each sideof the optic nerve in the horizontal and vertical direction). This areawas imaged with 1000 A-scans from the retinal pigment epithelium (RPE)to the posterior lens and 100 B-scans across the selected area at 2×2×2micron voxels. Thus, approximately one third of the central retina ofeach mouse was imaged. In addition, images from other areas wereregularly obtained to confirm that the central area was representativeof the whole retina.

B scans through the rectangular scanned area are displayed as a seriesof retinal cross sections as would be seen in a light microscope whenviewing histological sections taken horizontally through the retina(FIG. 3). The scans also provide an enface image of the whole areascanned (volume intensity projection) similar to fundoscopic images.Cavities consist of abnormal separations of retinal tissue between andwithin the outer plexiform (OPL) and inner nuclear layers (INL) (Figure.3). Though the resolution is less than in microscopic images, theindividual retinal layers and histopathology of Rs1-KO retinas can beeasily distinguished. As imaged by OCT in mice, these cavities extendedtens to hundreds of microns in radial length (optic nerve to periphery)and several microns to tens of microns in axial depth (across retinalthickness). They have an appearance very similar to that seen in fixedtissue under the microscope. In the volume intensity projections, thedistribution of cavities within the scanned area is seen as patches oflight and dark.

FIG. 3 shows OCT scans from a wild type and an Rs1-KO mouse showing aB-scan (left-hand images) taken through the central retina at the opticnerve as indicated by the central green line on the volume intensityprojection (right-hand images) for each eye. The volume intensityprojection is similar to a fundus photo imaging the surface of theretina from the front of the eye. The layers in the retina of the WT arewell organized and distinct and the fundus has a smooth appearance anddistinct retinal vessels. The B-scan of the Rs1-KO retina shows largeareas of separations, called “schisis cavities,” and the layers are lessorganized, less distinct and thinner. The fundus has a mottledappearance. Red asterisks indicate location of measurements of cavitywidth. Each measurement was graded on a scale of one to six, and thesmallest and largest of these six measurements were averaged to producea score for each retina.

The maximum height of these separations or “schisis cavities” in theinner nuclear layer of treated and untreated Rs1-KO retinas was measuredalong the B scans in four separate areas: one measurement 0.6 mmsuperior to the optic nerve, one measurement on the nasal and one on thetemporal side of the midline scan through the optic nerve, and onemeasurement 0.6 mm inferior to the optic nerve as indicated by the redasterisks in FIG. 3 using an onscreen micrometer. The measurements werecombined to generate a score for each retina as follows:

Data Collection

-   -   1. The central one-third of the retinal area was scanned by OCT        for retinal cavities. Determination of the retinal area to be        scanned was based on the following considerations:        -   a. In a previously published study, we found that retinal            pathology in the form of cavities was maximal in number and            extent between 1 and 4 months of age and were distributed            from optic nerve to periphery.        -   b. Post injection times of 11 to 15 weeks in the present            study, meant animals would be analyzed by OCT when untreated            eyes of Rs1-KO mice would have maximal cavity number and            distribution.        -   c. Vector was injected in the center of the vitreous and            assumed to distribute equally in all directions.        -   d. The central one-third of the retina OCT imaged by a            single rectangular scan in Rs1-KO mice was representative of            the rest of the retina.    -   2. Linear scans were done at position A (+0.6 mm), B (0 mm or        optic nerve), and C (−0.6 mm) (FIG. 3, Rs1-KO, right-hand image)

Cavity Grading

-   -   Three scans were performed: scan A and C at the most superior        and inferior extent, respectively, were graded for maximum        cavity height; scan C through the central retina was graded for        maximum cavity height on each side of midline: total 4 values        for each retina (asterisks in FIG. 3):        -   a. No cavities=1        -   b. Cavities <30 μm in height=2        -   c. Cavities 30 to 49 μm in height=3        -   d. Cavities 50 to 69 μm in height=4        -   e. Cavities 70 to 99 μm in height=5        -   f. Cavities >100 μm in height=6

Scoring Formula:

Score=(maximum grade at position 1-4+minimum grade at position 1-4)/2.

Rs1 Protein Expression by Immunohistochemistry

The retinas of all animals that survived one or two ERG recordings weretaken for retinal retinoschisin immunostaining to quantify retinoschisinexpression.

From 1 to 21 days after the ERG or OCT the mice were euthanized andperfused with 4% paraformaldehyde in sodium phosphate buffer. The eyeswere removed and fixed overnight in 4% paraformaldehyde and 0.5%glutaraldehyde in sodium phosphate buffer followed by processing forcryosectioning. Twenty-five sagittal sections of the injected eye weretaken beginning at the nasal margin of the retina and proceeding throughand including the optic nerve head and approximately 200 μm of thetemporal retina. The sections were stained using a rabbit polyclonalantibody against the N-terminus of retinoschisin (amino acid residues24-37) and a secondary antibody conjugated to red-fluorescent AlexaFluor 568 dye (Invitrogen). Nuclei were stained with DAPI.

Retinoschisin expression in retinas of eyes receiving AAV8 scRS/IRBP hRSand untreated eyes was evaluated using a fluorescence microscope todetermine the intensity and extent of immunostaining in 4 verticalsections taken at evenly spaced intervals from the nasal margin of theretina to the optic nerve and one section taken just temporal to theoptic nerve. The results from these 5 sections were averaged. In eachsection from a vector treated Rs1-KO retina, stain intensity in thephotoreceptor and inner retinal layers was evaluated in comparison to aWT retina stained at the same time to help control for variations in thelevel of background staining with each batch. WT intensity was assigneda value of 4, and Rs1-KO sections were graded from 0 to 7 as depicted inFIG. 4A. FIG. 4 shows the scoring of retinoschisin immunostaining inAAV8 scRS/IRBP hRS treated retinas of Rs1-KO mice. Retinoschisin proteinwas visualized by immunofluorescent labeling (red) in frozen retinalsections from wild type (WT) mice and Rs1-KO mice treated with AAV8scRS/IRBP hRS by intravitreal injection. Retinas were processed 12 to 18weeks after injection. Scoring of retinoschisin staining was done usingintensity and distribution grading. FIG. 4A shows the intensity criteriafor levels 0-7 are shown. Levels 0 through 4 could be graded on thebasis of photoreceptor staining intensity only (solid white arrows)because photoreceptor staining was not saturated. At level 4 innerretinal staining is also seen. Level 4 staining in an Rs1-KO and WTretina are shown. Since photoreceptor staining approaches saturation atlevel 4, levels 5, 6 and 7 are graded on the basis of staining intensityand consistency in the inner nuclear (open white arrow) and innerplexiform (white bar) layers. An increase in brightness and/or moresolid staining than the previous grade in either layer was used ascriteria for the next highest grade. In grade 5 above, the IPL wasstained more intensely than in grade 4; in grade 6, the IPL was similarto grade 5, but the INL was brighter than 5; in grade 7, the IPL wasstained more solidly than in grade 6 Staining in the RPE layer (oval inRs1-KO and WT #4) when present was ignored. FIG. 4B shows the method ofcombining staining intensity with distribution to obtain staining score.The proportion of the section stained was defined by the widestseparation of stained tissue (not necessarily uninterrupted). Thestrongest and weakest staining grades within that length were addedtogether and multiplied by the proportion of the sectioned stained toobtain the staining score. Examples are high levels of expression inretinas treated with three different doses of scAAV8/IRBP hRS.

Since retinoschisin staining was not uniformly distributed across thesections from most treated retinas, two scores for each section wereused: one score was assigned to the weakest area, and another one wasassigned to the strongest area. The lowest and highest grades in eachretina were added together. If staining was consistent across thesection, the grade was doubled. Sections from WT retinas were uniformlystained and so had an intensity grade of 8 (4+4). Since in many sectionsstaining was limited to only a portion of the retinal length, thestaining intensity grade was multiplied by the proportion of the retinallength over which staining was observed to obtain the final score (FIG.4B). For example, if staining intensity in a section ranged from 1 to 8but only ½ the retinal section had retinoschisin staining, the scorewould be (1+8)*½=4.5. Wild type retinas received a score of 8:(4+4)*1/1. None of the untreated eyes showed staining above non-specificbackground levels (a score of 0).

Immunostaining Score Formula:

Score per section=(maximum staining grade in section[0-7]+minimumstaining grade in section)×(proportion of entire length containing allstaining). Score per eye=(Score for sections 1+2+3+4+5)/5.

Results ERG, Oct and Retinoschisin Expression Analysis

In this study, vehicle or AAV8 scRS/IRBP hRS vector at doses of 1.0e6,1.0e7, 5.0e7, 1.0e8, 5.0e8 and 2.5e9 vg/eye, were administered byintravitreal injection to 18-34 day old Rs1-KO mice. The mice were thenevaluated by ERG for retinal function at 11-15 weeks and 6-9 months PIfollowed by OCT for retinal structure and immunohistochemistry forretinoschisin expression. These experiments were conducted to determinethe dose range over which this vector significantly preserves retinalfunction and structure in the Rs1-KO mouse and achieves significantretinal expression of protein. FIGS. 5-9 show the ERG, OCT (retinalcavity) and retinoschisin expression data.

FIG. 5 shows the ERG a- and b-wave amplitudes of untreated eyes and eyestreated with intravitreal injections of vehicle or AAV8 scRS/IRBP hRSvector at doses of 1e6, 1e7, 5e7, 1e8, 5e8 and 2.5e9 vg/eye. 18-34 dayold Rs-1KO mice received vehicle or AAV8 scRS/IRBP hRS vector in one eyeby intravitreal injection at the doses indicated (expressed as vectorgenomes/eye). Amplitude values for a- and b-waves are shown and thegroup sizes are given below the dose. Two, 3 and 4 stars (*) indicatetreated values that differ from untreated values, with P values <0.01,0.001, and 0.0001, respectively, based on the unpaired t test correctedfor multiple comparisons using the Holm-Sidak method. These eyes wereevaluated between 11 and 15 weeks, post injection, a time period deemed“Short Term.”

FIG. 6 shows the ERG a- and b-wave amplitudes in animals receiving 1e8,5e8 and 2.5e9 vg/eye vector doses at 6-9 months post injection, a timeperiod deemed “Long Term”. This group is a subset of the animalsevaluated at the Short Term time point in FIG. 5.

FIG. 7 presents a comparison of the Short Term and Long Term ERG resultsfor treated and untreated eyes at vector doses of 1e8, 5e8 and 2.5e9vg/eye, so that persistence of treatment efficacy can be evaluated. Thisdata is derived from the data sets in panels FIGS. 5 and 6. The lines atthe top of the bars were drawn to allow the reader to better visualizethe changes over time. One, 2, and 4 stars (*) indicate treated valuesthat differ from untreated values, with P values <0.05, 0.01, and0.0001, respectively, based on the unpaired t test corrected formultiple comparisons using the Holm-Sidak method.

FIG. 8 shows the schisis cavity scoring averages in treated anduntreated eyes from OCT images for vector doses of 1e6, 1e7, 5e7, 1e8,5e8 and 2.5e9 vg/eye. The mice were evaluated at the Short Term timepoint (11-15 weeks post-injection). The bar graph shows the average rawscores (±SEM) for treated and untreated retinas at each dose andstatistical comparison of treated to untreated eye based on the unpairedt test corrected for multiple comparisons using the Holm-Sidak method.The mice were evaluated at the Short Term time point (11-15 weekspost-injection) directly after ERG recording (data in panel A).(**P<0.01, **** P<0.0001).

FIG. 9 depicts retinoschisin protein expression in response to vectordoses between 1e7, 1e8, 5e8, and 2.5e9 vg/eye. The mice were examined atthe Short Term time point (11-15 weeks, post injection). The 2.5e9vg/eye dose was also evaluated at the Long Term time point (6-9 months,post-injection). Scatter plot shows values for each animal, the meansand 95% confidence interval for each dose and statistical comparison tountreated eyes, by the one sample t test. (*** P<0.001, **** P<0.0001).

CONCLUSIONS

In this study, vehicle or AAV8 scRS/IRBP hRS vector at doses of 1.0e6,1.0e7, 5.0e7, 1.0e8, 5.0e8 and 2.5e9 vg/eye, were administered byintravitreal injection to 18-34 day old Rs1-KO mice. The mice were thenevaluated by ERG for retinal function at 11-15 weeks and 6-9 months PIfollowed by OCT for retinal structure and immunohistochemistry forretinoschisin expression. The experiments were designed to determine thedose range over which this vector significantly preserves retinalfunction and structure in the Rs1-KO mouse and achieves significantretinal expression of protein. From these experiments the following canbe concluded:

-   -   1. AAV8 scRS/IRBP hRS vector doses of 5e7, 1e8 and 2.5e9 vg/eye        showed statistically significant improvement in ERG a-wave        amplitudes, and doses of 5e7, 1e8, 5e8, or 2.5e9 vg/eye showed        statistically significant improvement in ERG b-wave amplitudes,        compared to uninjected eyes, when recorded 11-15 weeks,        post-injection (Short Term time point). Injection vehicle had no        effect.    -   2. Vector doses of 1e8, 5e8 and 2.5e9 vg/eye produced        statistically significant improvement in ERG a- and b-wave        amplitudes compared to untreated eyes, when recorded 6-9 months,        post injection (Long Term time point).    -   3. Vector doses of 5e7, 1e8, 5e8 and 2.5e9 vg/eye produce        statistically significant improvement in schisis cavity scoring        relative to untreated eyes when evaluated following the 11-15        weeks post-injection (Short Term time point).    -   4. Retinoschisin protein expression is significantly elevated in        Rs-1/KO mouse eyes after vector treatment with doses of 1e7,        1e8, 5e8, and 2.5e9 vg/eye evaluated following the 11-15 weeks,        post-injection (Short Term time point). At vector doses of 1e8        vg/eye and above, retinoschisin protein expression is greater        than or equal to 25% of wild type levels. The Rs-1/KO eyes        receiving the 2.5e9 vg/eye dose were evaluated at 6-9 months        post-injection and showed 65% of the wild type retinoschisin        level. This was the only dose evaluated at the Long Term time        point.

Eyes of Rs-1/KO mice treated with AAV8 scRS/IRBP hRS vector dosesbetween 1e8 and 2.5e9 vg/eye show statistically significant improvementin retinal function (by ERG) and retinal structure (by OCT) and expresssignificant amounts of retinoschisin protein. When scaled to humans byretinal surface area (factor of 100), these doses are 1e10 to 2.5e11vg/eye. In the accompanying Toxicity Report, rabbits treatedintravitreally with AAV8 scRS/IRBP hRS vector at a dose of 2.5e10 vg/eyeshowed little toxicity at 4 months post injection.

Example 2

A study was conducted to assess the tolerability of expression vectorsof the invention in rabbit eyes compared with control injections ofvehicle alone. Briefly, thirty-nine New Zealand White rabbits (age 6-7months at injection; weight 2.4-3.8 kg) were used in the present study.All in life procedures were conducted in compliance with the ARVOStatement for the Use of Animals in Ophthalmic and Vision Research andwere approved by the Animal Care and use Committee of the National EyeInstitute.

Vectors or vehicle were administered in the right eye by intravitrealinjection using ½ cc Insulin Syringes with permanently attached 28 gaugeneedle (Ultrafine U-100 syringe—BD Biosciences, San Jose, Calif.) in aninjection volume of 50 ul. Syringes were loaded under sterile conditionsin a laminar flow hood on the day of injection. Rabbits wereanesthetized with IM ketamine, 40 mg/kg, and xylazine, 3 mg/kg. Sterilesurgical instruments were used and the animals were prepared asepticallyprior to injection. Povidone iodine (5% povidone iodine, 95%BSS-irrigating solution) was used to disinfect the eyelid margins andeye lashes. BSS solution was used to wash the eyelids and for eyeirrigation every 2 minutes to minimize corneal air exposure andconsequent abrasion. An eyelid speculum was applied to avoidmanipulating the eye and to avoid needle contact with lids and lashes.Fifty microliters of vector or vehicle solution were injected throughthe pars plana in the superior temporal quadrant approximately 2 mmposterior to the limbus in each eye. The injection was performed withthe needle tip in the center of the vitreous and the vector wasdelivered at a moderately slow rate. The needle was then carefullyextracted from the eye and a sterile cotton-tip applicator was appliedto prevent reflux of both the vector and vitreous. Triple antibioticophthalmic ointment (“neo-poly-bac” for neomycin, polymixin andbacitracin) was applied to the injection site after injection, and therabbits were returned to their cages.

All rabbits were clinically examined before injection, at 14 days and 1,2 and 3 months after injection. Each rabbit underwent external ocularinspection and full ocular examination by slit lamp biomicroscopy(anterior segment) and by indirect ophthalmoscope (posterior segment)after pupillary dilation (one drop—twice, 10 minutes apart of topicalAtropine 1% in both the eyes). Clinical changes were graded using a5-step severity scale (none, trace, +1, +2, +3) by two examiners whowere blinded to the nature or dose of the treatment.

The results from this study are shown in FIG. 10. Findings from both theinjected (Inj.) and uninjected eye (Uninj) are displayed for each animalat five time points (0, 2, 4, 8, 12 weeks) during the study. (n=4-7rabbits/group). The data demonstrates that at two high doses (2 e¹⁰vg/eye or 2 e¹¹ vg/eye) of the expression vector injected into therabbit eyes, only minimal inflammation post-injection (trace or mild)was detected in a few test animals. Mild inflammation resolved in mostanimals at both dosages over time.

The foregoing examples of the present invention have been presented forpurposes of illustration and description. Furthermore, these examplesare not intended to limit the invention to the form disclosed herein.Consequently, variations and modifications commensurate with theteachings of the description of the invention, and the skill orknowledge of the relevant art, are within the scope of the presentinvention. The specific embodiments described in the examples providedherein are intended to further explain the best mode known forpracticing the invention and to enable others skilled in the art toutilize the invention in such, or other, embodiments and with variousmodifications required by the particular applications or uses of thepresent invention. It is intended that the appended claims be construedto include alternative embodiments to the extent permitted by the priorart.

What is claimed is: 1-69. (canceled)
 70. An expression vector comprisingan expression cassette, wherein the expression cassette comprises apromoter operably-linked to a nucleic acid sequence encoding atherapeutic protein, wherein the level of expression of the therapeuticprotein is high enough that the amount of a delivery vehicle comprisingthe expression vector needed to alleviate symptoms of an eye disease,elicits a minimal immune response when administered to the eye.
 71. Theexpression vector of claim 70, wherein the promoter is an eye-specificpromoter.
 72. The expression vector of claim 70, wherein the promotercomprises at least a portion of a promoter selected from the groupconsisting of a retinoschisin promoter, a rhodopsin promoter, arhodopsin kinase promoter, a CRX promoter, and an interphotoreceptorretinoid binding protein (IRBP) promoter
 73. The expression vector ofclaim 70, wherein the expression cassette comprises an enhancer sequencethat enhances the activity of the promoter.
 74. The expression vector ofclaim 73, wherein the enhancer sequence is an interphotoreceptorretinoid binding protein enhancer sequence.
 75. The expression vector ofclaim 70, wherein the expression cassette comprises at least a portionof intron 1 of a retinoschisin gene.
 76. The expression vector of claim75, wherein the at least a portion is located within the nucleic acidsequence encoding a therapeutic protein.
 77. The expression vector ofclaim 75, wherein the at least a portion of intron 1 comprises thesplice donor and splice acceptor sequences of intron 1 of aretinoschisin gene.
 78. The expression vector of claim 70, wherein thetherapeutic protein is selected from the group consisting of aretinoschisin protein, ciliary neurotropic factor (CNTF), brain-derivedneurotropic factor (BDNF), pigment epithelium-derived factor (PEDF). 79.The expression vector of claim 70, wherein the expression cassette isflanked by adeno-associated virus inverted terminal repeats (ITRs). 80.The expression vector of claim 79, wherein at least one of the ITRs hasbeen modified at the rep nicking site or within the D region.
 81. Aviral vector comprising an expression cassette, wherein the expressioncassette comprises a promoter operably-linked to a nucleic acid sequenceencoding a therapeutic protein, wherein the level of expression of thetherapeutic protein is high enough that the amount of a delivery vehiclecomprising the expression vector necessary to alleviate symptoms of aneye disease, elicits a minimal immune response when administered to theeye.
 82. The viral vector of claim 81, wherein the promoter is aneye-specific promoter.
 83. The viral vector of claim 81, wherein theexpression cassette comprises an enhancer sequence that enhances theactivity of the promoter.
 84. The viral vector of claim 81, wherein theexpression cassette comprises at least a portion of intron 1 of aretinoschisin gene.
 85. The viral vector of claim 81, wherein thetherapeutic protein is selected from the group consisting of aretinoschisin protein, ciliary neurotropic factor (CNTF), brain-derivedneurotropic factor (BDNF), pigment epithelium-derived factor (PEDF). 86.The viral vector of claim 81, wherein the viral vector comprises acapsid protein from an adeno-associated virus (AAV).
 87. A method fortreating an eye disease, the method comprising administering to anindividual in need of such treatment an expression vector comprising anexpression cassette, wherein the expression cassette comprises apromoter operably-linked to a nucleic acid sequence encoding atherapeutic protein, wherein the level of expression of the therapeuticprotein is high enough that the amount of a delivery vehicle comprisingthe expression vector necessary to alleviate symptoms of an eye disease,elicits a minimal immune response when administered to the eye.
 88. Themethod of claim 87, wherein the therapeutic protein is a retinoschisinprotein and the eye disease is X-linked retinoschisis.
 89. The method ofclaim 87, wherein the expression vector is administered by a routeselected from the group consisting of intravitreally, subretinally,subtenonly and subconjuntivally.